CN110551217B - IL-11R binding proteins and uses thereof - Google Patents

IL-11R binding proteins and uses thereof Download PDF

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CN110551217B
CN110551217B CN201910868161.5A CN201910868161A CN110551217B CN 110551217 B CN110551217 B CN 110551217B CN 201910868161 A CN201910868161 A CN 201910868161A CN 110551217 B CN110551217 B CN 110551217B
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K·M·爱德华兹
M·P·哈迪
V·瑞兹曼
M·威尔森
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CSL Ltd
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Abstract

The present application provides proteins comprising an antigen binding site of an antibody that binds interleukin-11 (IL-11) receptor alpha (IL-11 ra) and uses thereof, for example in therapy.

Description

IL-11R binding proteins and uses thereof
Information of related application
The present application claims priority from australian patent application No. 2013900389 entitled "IL-11R binding protein and use thereof" filed on 7 th 2 nd 2013 and from U.S. patent application No. 61/764,756 entitled "IL-11R binding protein and use thereof" filed on 14 nd 2 nd 2013. The entire contents of these documents are incorporated herein by reference.
Sequence listing
The application is attached with a sequence table submitted in an electronic format. The entire contents of this sequence listing are incorporated herein by reference.
Technical Field
The present application relates to proteins comprising an antigen binding site of an antibody that binds interleukin-11 (IL-11) receptor alpha (IL-11 ra) and uses thereof, for example in therapy.
Background
IL-11 is a member of the IL-6 cytokine family, which also includes IL-27, IL-31, leukemia Inhibitory Factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and the like. IL-6 family cytokines induce signal transduction via the common signal transduction receptor beta subunit gp130 and the specific receptor alpha subunit. For IL-11, binding of this cytokine to its specific receptor alpha subunit IL-11Rα induces gp130 homodimerization. Dimerization of gp130 activates the JAK/STAT signaling pathway and results in activation of the signal transduction and transcriptional activator (STAT) 3 (STAT 3) and to a lesser extent STAT1.
IL-11 signaling is known to play a role in hematopoiesis, immune response, inflammation, adipogenesis, osteoclast formation, neurogenesis, megakaryocyte maturation, and platelet production. IL-11 is clinically used or is being developed for the treatment of a variety of conditions, such as chemotherapy-induced thrombocytopenia and a variety of inflammatory diseases, including arthritis, inflammatory bowel disease, radiation-induced lung injury, sepsis and psoriasis. However, the clinical use of IL-11 is limited because serious adverse events (including oedema) are reported. Furthermore, IL-11 has been shown to have deleterious effects in a variety of conditions.
For example, IL-11 has been found to act as an inhibitor of bone formation, and is critical for osteoclast formation and activity, as well as bone resorption. Thus, it has been proposed to treat osteoporosis and prevent bone resorption/promote bone formation in other conditions (such as metastatic bone cancer, myeloma, paget's disease, and bone fracture and healing) by blocking the activity of IL-11.
IL-11 signaling has been shown to have pathogenic effects in the early stages of tuberculosis. Blocking IL-11 with anti-IL-11 antibodies was shown to impair histopathology of lung tissue and neutrophil infiltration in mycobacterium tuberculosis infected mice.
Antagonism of IL-11 is also proposed as a method of treating Th2 mediated diseases including asthma, chronic Obstructive Pulmonary Disease (COPD), rhinitis, allergy and atopic dermatitis. In this regard, blocking IL-11 signaling using mutant forms of IL-11 that do not induce signaling is shown to have therapeutic benefit in a mouse asthma model.
IL-11 and/or IL-11Rα are overexpressed in liver cancer, pancreatic cancer, gastric cancer, osteosarcoma, endometrial cancer, and ovarian cancer. Furthermore, as described above, IL-11 induced gp130 dimerization leads to activation of STAT3, which induces expression of angiogenesis-related genes (e.g. VEGF), cell cycle progression-related genes (e.g. cyclin D1) and cell survival-related genes (e.g. Bcl-XL, survival). Sustained STAT3 activity appears to be associated with hematological malignancies and tumors of epithelial origin. Excessive STAT3 activation promotes the growth and survival of gastric cells, associated with increased gastric angiogenesis and leads to gastric tumorigenesis in mice. However, gastric inflammation, proliferation and tumor formation were inhibited in IL-11 non-responsive mice or mice treated with non-signal transduction mutant variants of IL-11.
IL-11 is also involved in other biological processes such as inhibition of adipogenesis, induction of cachexia (e.g., cancer cachexia), induction of fever, regulation of extracellular matrix metabolism, stimulation of acute phase reactants, and embryo transfer.
It should be appreciated by those skilled in the art from the foregoing that agents that neutralize IL-11 signaling are needed because of their potential to provide therapeutic benefits in any of a wide variety of conditions. There is also a need for agents that bind IL-11 ra because of their advantage of being able to specifically target cells in vivo without the need to bind and neutralize dissolved IL-11 in the subject.
Despite this need, many agents that bind IL-11Rα (e.g., antibodies) do not neutralize IL-11 signaling. For example, blanc et al (Journal of Immunological Methods 241:43-59, 2000) describe a panel of 14 murine monoclonal antibodies raised against human IL-11Rα, but none of them was able to inhibit IL-11-induced proliferation of BaF3/gp130/IL-11R cells, indicating that these antibodies do not neutralize IL-11 signaling. Commercially available anti-IL-11 ra antibodies, such as 4D12 available from santa claus biotechnology company (Santa Cruz Biotechnology, inc.) also do not neutralize IL-11 signaling.
Disclosure of Invention
In producing the present invention, the inventors wish to produce agents (e.g., antibodies or proteins comprising antigen binding domains thereof) that bind to IL-11 ra and neutralize IL-11 signaling. The inventors produced a series of antibodies with such activity, some of which strongly neutralized IL-11 signaling, e.g., prevented IL-11 dependent BaF3 cell proliferation. These antibodies have been shown to cross-react with human IL-11Rα (hIL-11 Rα) and cynomolgus IL-11Rα (cynomolgus 11Rα), indicating their use in primate models of human disease. These antibodies were also found to bind overlapping epitopes. The inventors then affinity matured one of these antibodies and produced a series of other antibodies with other desirable properties, such as neutralizing IL-11 signaling and/or improved affinity and/or sequences similar to human germline (e.g., reduced likelihood of inducing an immune response when administered to a human).
Based on the foregoing, the following facts will be apparent to those skilled in the art: the inventors produced proteins comprising an antigen binding domain of an antibody that is capable of binding or specifically binding to IL-11 ra and neutralizing IL-11 signaling.
In one embodiment, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling, the antigen binding domain being capable of binding to both hIL-11Rα and cynoIL-11Rα.
In one embodiment, the IL-11Rα binding protein neutralizes human IL-11 (hIL-11) and/or cynomolgus IL-11Rα (cynoiIL-11 Rα) signaling.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds to IL-11Rα and neutralizes IL-11 signaling and inhibits IL-11 (e.g., hIL-11 or cynoIL-11) -mediated proliferation of BaF3 cells expressing IL-11Rα and gp130, the IC thereof 50 Is 10 μg/ml or less. In one embodiment, the IC 50 Is 5 μg/ml or less. For example, the IC 50 4 μg/ml or less or 3.5 μg/ml or less. In one embodiment, the IC 50 3 μg/ml or less or 2 μg/ml or less. For example, the IC 50 Is 1g/ml or less. For example, the IC 50 0.9 μg/ml or less or 0.8 μg/ml or less or 0.7 μg/ml. In one embodiment, the IC 50 0.7 μg/ml or less. In one embodiment, in connection with each of the foregoing embodiments, the IC 50 May be 10pg/ml or more or 10ng/ml or more.
In one embodiment, the IC 50 10nM or less. For example, the IC 50 8nM or less or 7nM or less. In one embodiment, the IC 50 6nM or less or 6.5nM or less. For example, the IC 50 Is 5nM or less. For example, the IC 50 4.5nM or less. For example, the IC 50 Is 4nM or less. In one embodiment, in connection with each of the foregoing embodiments, the IC 50 May be 10pM or higher.
In one embodiment, the IL-11Rα binding protein inhibits IL-11 (e.g., hIL-11 or cynoIL-11) mediated proliferation of BaF3 cells expressing IL-11Rα and gp130, IC 50 Is at least about 1.5 times that of antibody 8E2 (the heavy chain of which comprises the sequence set forth in SEQ ID NO:83 and the light chain of which comprises the sequence set forth in SEQ ID NO: 84). In one embodiment, the IC 50 At least about 2-fold or at least about 2.5-fold or at least about 3-fold of antibody 8E 2.
In one embodiment, the IL-11Rα binding protein inhibits IL-11 (e.g., hIL-11) mediated proliferation of BaF3 cells expressing IL-11Rα and gp130, IC 50 At least about 1.5-fold (i.e., about 1.5-fold lower IC50 value) or 2-fold or 3-fold, of an antagonistic hIL-11 mutein (e.g., comprising the sequence set forth in SEQ ID NO: 110), wherein IC 50 Measured in nM. In one embodiment, the IC 50 At least about 3.5-fold or at least about 4-fold of the mutein.
In one embodiment, the method is performed by contacting the cell surface in the presence of about 0.3ng/mL hIL-11 to about 5ng/mL hIL-11 (e.g., in the presence ofAbout 0.3ng/mL hIL-11 or about 0.5ng/mL hIL-11 or about 5ng/mL hIL-11 in the presence of BaF3 cells expressing IL-11Rα and gp130 (e.g., genetically modified to express IL-11Rα and/or gp 130) (e.g., about 1x 10) 4 Individual cells) for about 48-50 hours or about 48 hours or about 50 hours to determine IC 50 . In one embodiment, the cells are cultured in the presence of the protein of the invention (e.g., for about 30 minutes) prior to addition of IL-11. In one embodiment, proliferation is determined by measuring 3H-thymidine incorporation into DNA during the last 6 hours of incubation. In an assay for determining neutralization of cynoIL-11, these cells can be cultured in the presence of about 0.5ng/mL cynoIL-11 to about 5ng/mL cynoIL-11 (e.g., in the presence of about 0.5ng/mL cynoIL-11 or about 5ng/mL cynoIL-11).
Additionally or alternatively, the invention provides an IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds to IL-11 ra and neutralizes IL-11 signaling and which IL-11 ra binding protein hybridizes to SEQ ID NO:86 polypeptide is less than the binding level of the IL-11 ra binding protein to SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds to IL-11 ra and neutralizes IL-11 signaling and which IL-11 ra binding protein hybridizes to SEQ ID NO:89 polypeptide is lower than the binding level of the IL-11 ra binding protein to SEQ ID NO:3 and/or 85 polypeptide.
In one embodiment, the level of binding is determined by Western blotting and/or by Fluorescence Activated Cell Sorting (FACS) of cells expressing the polypeptide.
In one embodiment, the IL-11Rα binding protein binds to the sequence of SEQ ID NO:3 and/or 85 polypeptide, and the IL-11 ra binding protein hybridizes to SEQ ID NO: the level of binding of the 86 or 89 polypeptide is reduced by at least about 10-fold or 20-fold or 50-fold or 100-fold or 150-fold or 200-fold.
In one embodiment, the IL-11Rα binding protein binds to the sequence of SEQ ID NO:86 or 89 polypeptide.
In one embodiment, the IL-11Rα binding protein binds to the sequence set forth in SEQ ID NO:87 or 88 polypeptide. For example, the IL-11Rα binding protein hybridizes to SEQ ID NO:87 or 88 polypeptide and the IL-11Rα binding protein and SEQ ID NO:3 and/or 85 polypeptide is similar or substantially the same (e.g., within about 20% or 15% or 10% or 5%).
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and that binds an epitope comprising residues within a first fibronectin type III domain of IL-11Rα.
In one embodiment, the epitope comprises residues of the first fibronectin type III domain and the immunoglobulin-like domain of IL-11Rα.
In one embodiment, the epitope comprises SEQ ID NO:1 between amino acids 111-215.
In one embodiment, the epitope comprises SEQ ID NO: residues 1-215 of amino acid 1.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8E2 (V thereof H Comprising SEQ ID NO:37 and V thereof L Comprising SEQ ID NO: 5) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8E4 (V thereof H Comprising SEQ ID NO:74 and V thereof L Comprising SEQ ID NO: 73) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody, which antigen binding domainThe binding domain binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8D10 (V thereof H Comprising SEQ ID NO:76 and V thereof L Comprising SEQ ID NO: 75) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8E2 (comprising a heavy chain comprising the sequence set forth in SEQ ID NO: 37V H And a human IgG4 constant region, the light chain comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L And human light chain constant region) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8E4 (comprising a heavy chain comprising the sequence V set forth in SEQ ID NO:74 H And a human IgG4 constant region, the light chain comprising SEQ ID NO: 73V of the sequence set forth in SEQ ID NO. 73 L And human light chain constant region) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the protein competitively inhibits antibody 8D10 (comprising a heavy chain comprising the sequence V set forth in SEQ ID NO:76 H And a human IgG4 constant region, the light chain comprising SEQ ID NO: 75V of the sequence set forth in SEQ ID NO. 75 L And human light chain constant region) and hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and that competitively inhibits antibody 8E2 (whose heavy chain comprises the sequence set forth in SEQ ID NO:83 and whose light chain comprises the sequence set forth in SEQ ID NO: 84) from binding to hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and that competitively inhibits antibody 8E4 (whose heavy chain comprises the sequence set forth in SEQ ID NO:92 and whose light chain comprises the sequence set forth in SEQ ID NO: 91) from hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and that competitively inhibits antibody 8D10 (whose heavy chain comprises the sequence set forth in SEQ ID NO:94 and whose light chain comprises the sequence set forth in SEQ ID NO: 93) from binding to hIL-11Rα and/or SEQ ID NO:3 and/or 85 polypeptide.
Additionally or alternatively, the invention provides an IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds to IL-11 ra and neutralizes IL-11 signaling and which IL-11 ra binding protein hybridizes to SEQ ID NO: the binding level of the 95 polypeptide is lower than that of the IL-11Rα binding protein and the sequence of SEQ ID NO:85 binding level of the polypeptide.
Additionally or alternatively, the invention provides an IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds to IL-11 ra and neutralizes IL-11 signaling and which IL-11 ra binding protein hybridizes to SEQ ID NO:96 polypeptide is less than the binding level of the IL-11 ra binding protein to SEQ ID NO:85 binding level of the polypeptide.
In one embodiment, the IL-11Rα binding protein has a reduced level of binding to a polypeptide comprising a substitution of at least about 1.5-fold or 2-fold.
In one embodiment, the IL-11Rα binding protein has a reduced level of binding to a polypeptide comprising a substitution of at least 3-fold or 4-fold or 5-fold or 10-fold.
In one embodiment, the IL-11Rα binding protein does not detectably bind to a polypeptide comprising a substitution.
In one embodiment, the level of binding is assessed using a biosensor (e.g., by surface plasmon resonance). For example, IL-11Rα binding protein is immobilized and assayed for binding to SEQ ID NO: 85. binding levels of 95 or 96 polypeptides. As exemplified herein, affinity can be determined by assessing the level of binding at several concentrations.
In another embodiment, the binding level is assessed using FACS. For example, the IL-11Rα binding protein hybridizes to a polypeptide that expresses SEQ ID NO: 85. 95 or 96 polypeptide or a form comprising a substituted IL-11 ra as described herein.
In one embodiment, the sequence that binds to SEQ ID NO:95 or 96, the IL-11 ra binding protein preferentially binds to SEQ ID NO:85 polypeptide.
In one embodiment, the sequence that binds to SEQ ID NO:95, the IL-11 ra binding protein preferentially binds to SEQ ID NO:85 polypeptide.
In one embodiment, the IL-11Rα binding protein competitively inhibits antibody 8E2 (V thereof H Comprising SEQ ID NO:37 and V thereof L Comprising SEQ ID NO: 5) and/or 8E4 (V thereof H Comprising SEQ ID NO:74 and V thereof L Comprising SEQ ID NO: 73) and/or 8D10 (V thereof H Comprising SEQ ID NO:76 and V thereof L Comprising SEQ ID NO: 75) and SEQ ID NO:3 and/or 85 polypeptide.
In one embodiment, the antigen binding domain cross-reacts with:
(i) SEQ ID NO: a 97 polypeptide; and/or
(ii) SEQ ID NO:98 polypeptide; and/or
(iii) SEQ ID NO:99 polypeptide.
In one embodiment, the IL-11Rα binding protein does not detectably bind to mouse IL-11Rα (SEQ ID NO:82 or 102) and/or SEQ ID NO:90 polypeptide.
In one embodiment, the IL-11Rα binding protein binds to mouse IL-11Rα (SEQ ID NO:82 or 102) and/or SEQ ID NO:90 polypeptide cross-reactions.
In one embodiment, the IL-11Rα binding protein is specific for human IL-11Rα and/or SEQ ID NO: affinity constant (K) for 3 and/or 85 polypeptides D ) Is about 9x10 -9 M or lower. For example, the K D Is about 8x10 -9 M or less or about 7x10 -9 M or less or about 6x10 -9 M or less or about 5x10 -9 M or lower. In one embodiment, the K D Is about 4.8x10 -9 M or lower.
In another embodiment, the IL-11Rα binding protein is specific for human IL-1iRα and/or the polypeptide of SEQ ID NO: affinity constant (K) for 3 and/or 85 polypeptides D ) Is about 2x10 -9 M or lower. For example, the K D Is about 1x10 -9 M or less or about 9x10 -10 M or less or about 8x10 -10 M or less or about 7x10 -10 M or lower. In one embodiment, the K D Is about 5x10 -10 M or lower. In one embodiment, the K D Is about 4x10 -10 M or lower. In one embodiment, the K D Is about 3x10 -10 M or lower. In one embodiment, the K D Is about 2x10 -10 M or lower. In one embodiment, the K D Is about 1.5x10 -10 M or lower.
In one embodiment, in connection with each of the preceding embodiments, the K D May be 0.1x10 -12 M or higher or 1x10 - 12 M or higher.
In one embodiment, K is evaluated using a biosensor (e.g., via surface plasmon resonance) D . For example, IL-11Rα binding protein is immobilized and assayed for binding to SEQ ID NO:85 binding level of the polypeptide.
In one embodiment, the IL-11Rα binding protein has a thermal transition midpoint (Tm) of about 60℃or greater. For example, the Tm is about 61 ℃ or greater, such as about 62 ℃ or greater, such as about 63 ℃ or greater. For example, the Tm is about 65℃or higher. For example, the Tm is about 69℃or higher. For example, the Tm is about 70℃or higher. Proteins with higher Tm are considered more stable, providing better storage and/or reducing effects after administration, e.g. due to aggregation.
Additionally or alternatively, the invention provides an IL-11 ra binding protein comprising an antigen binding domain of an antibody, the antigen binding domain binding or specifically binding to IL-11 ra and neutralizing IL-11 signaling and the antigen binding domain comprising one of:
(i) V comprising Complementarity Determining Regions (CDR) 1, CDR2 and CDR3 H The CDR1 comprises a sequence identical to SEQ ID NO:37 (or a sequence that is at least about 40% identical (or 50% identical or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence set forth between amino acids 31-35), said CDR2 comprises a sequence that is identical to SEQ ID NO:37 (or a sequence that is at least about 76% identical (or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) and the CDR3 comprises a sequence that is identical to SEQ ID NO:37 are at least about 55% identical (or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence set forth between amino acids 99-107;
(ii) Comprising a sequence identical to SEQ ID NO:37 or at least about 95% or 96% or 97% or 98% or 99% identical to V of the sequence set forth in seq id no H ；
(iii) V comprising CDR1, CDR2 and CDR3 L The CDR1 comprises a sequence identical to SEQ ID NO:5 (or 50% identical or 55% identical or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) and the CDR2 comprises the sequence set forth in SEQ ID NO:5 and the CDR3 comprises the sequence set forth between amino acids 50-56 of SEQ ID NO:5 (or a sequence that is at least about 44% identical (or 56% identical or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence set forth between amino acids 89-97;
(iv) Comprising a sequence identical to SEQ ID NO:5 (or a sequence that is at least about 94% identical (or 95% identical or 96% identical or 97% identical or 98% identical or 99% identical) in sequence) L ；
(v) V comprising CDR1, CDR2 and CDR3 H The CDR1 comprises SEQ ID NO:74, and said CDR2 comprises the sequence set forth between amino acids 31-35 of SEQ ID NO:74 and said CDR3 comprises the sequence set forth between amino acids 50-66 of SEQ ID NO:74 between amino acids 99-115;
(vi) Comprising SEQ ID NO: 74V of the sequence set forth in seq id no H ；
(vii) V comprising CDR1, CDR2 and CDR3 L The CDR1 comprises SEQ ID NO:73, and said CDR2 comprises the sequence set forth between amino acids 23-36 of SEQ ID NO:73 and the CDR3 comprises the sequence set forth between amino acids 52-58 of SEQ ID NO:73 between amino acids 91-101;
(viii) Comprising SEQ ID NO: 73V of the sequence set forth in SEQ ID NO. 73 L ；
(ix) V comprising CDR1, CDR2 and CDR3 H The CDR1 comprises SEQ ID NO:76, and said CDR2 comprises the sequence set forth between amino acids 31-35 of SEQ ID NO:76, and said CDR3 comprises the sequence set forth between amino acids 50-66 of SEQ ID NO:76 between amino acids 99-107;
(x) Comprising SEQ ID NO: 76V of the sequence set forth in seq id no H ；
(xi) V comprising CDR1, CDR2 and CDR3 L The CDR1 comprises SEQ ID NO:75, and said CDR2 comprises the sequence set forth between amino acids 24-34 of SEQ ID NO:75, and said CDR3 comprises the sequence set forth between amino acids 50-57 of SEQ ID NO:75 between amino acids 89-97 of 75;
(xii) Comprising SEQ ID NO: 75V of the sequence set forth in SEQ ID NO. 75 L ；
(xiii) V listed in (i) H And (iii) V L ；
(xiv) V listed in (i) H And (iv) V as set forth in L ；
(xv) V listed in (ii) H And (iii) V L ；
(xvi) V listed in (ii) H And (iv) V as set forth in L ；
(xvii) V listed in (V) H And (vii) V L ；
(xviii) V listed in (V) H And (viii) V L ；
(xix) V listed in (vi) H And (vii) V L ；
(xx) V listed in (vi) H And (viii) V L ；
(xxi) V listed in (ix) H And (xi) V L ；
(xxii) V listed in (ix) H And (xii) V L ；
(xxiii) V listed in (x) H And (xi) V L The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively
(xxiv) V listed in (x) H And (xii) V L 。
In one embodiment, the antigen binding domain comprises:
(i) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence listed between amino acids 31-35 of SEQ ID NO:37, CDR2 comprises a sequence that is at least about 80% identical (or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 50-66 of SEQ ID NO:37, and CDR3 comprises a sequence that is at least about 55% identical (or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 99-107 of SEQ ID NO: 37) and V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises the sequence set forth between amino acids 24-34 of SEQ ID NO:5, CDR2 comprises the sequence set forth between amino acids 50-56 of SEQ ID NO:5, and CDR3 comprises the sequence set forth between amino acids 89-97 of SEQ ID NO: 5) or comprises the sequence set forth in SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ii) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence listed between amino acids 31-35 of SEQ ID NO:37, CDR2 comprises a sequence that is at least about 80% identical (or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 50-66 of SEQ ID NO:37, and CDR3 comprises the sequence listed between amino acids 99-107 of SEQ ID NO: 37) and V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises the amino acid sequence of SEQ ID NO:5The sequences listed between residues 24-34, CDR2 comprises SEQ ID NO:5, and CDR3 comprises the sequence set forth between amino acids 50-56 of SEQ ID NO:5 between amino acids 89-97) or a sequence comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively
(iii) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence listed between amino acids 31-35 of SEQ ID NO:37, CDR2 comprises the sequence listed between amino acids 50-66 of SEQ ID NO:37, and CDR3 comprises a sequence that is at least about 55% identical (or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 99-107 of SEQ ID NO: 37) and V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises the sequence set forth between amino acids 24-34 of SEQ ID NO:5, CDR2 comprises the sequence set forth between amino acids 50-56 of SEQ ID NO:5, and CDR3 comprises the sequence set forth between amino acids 89-97 of SEQ ID NO: 5) or comprises the sequence set forth in SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L 。
In one embodiment, the antigen binding domain comprises:
(i) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence set forth between amino acids 31-35 of SEQ ID NO:37, CDR2 comprises the sequence set forth between amino acids 50-66 of SEQ ID NO:37, and CDR3 comprises the sequence set forth between amino acids 99-107 of SEQ ID NO: 37) or comprises the sequence set forth in SEQ ID NO:37, V of the sequence set forth in seq id no H V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises a sequence that is at least about 54% identical (or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 24-34 of SEQ ID NO:5, CDR2 comprises a sequence that is at least about 66% identical (or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence listed between amino acids 50-56 of SEQ ID NO:5, and CDR3 comprises a sequence that is at least about 66% identical (or 70% identical or 80% identical or 95% identical or 98% identical or 99% identical) to the sequence listed between amino acids 89-97 of SEQ ID NO: 5);
(ii) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence listed between amino acids 31-35 of SEQ ID NO:37 and CDR2 comprises SEQ ID NO: 37)Amino acids 50-66, and CDR3 comprises the sequence set forth in SEQ ID NO:37 between amino acids 99-107) or a sequence comprising SEQ ID NO:37, V of the sequence set forth in seq id no H V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises a sequence that is at least about 54% identical (or 60% identical or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence set forth between amino acids 24-34 of SEQ ID NO:5, CDR2 comprises a sequence set forth between amino acids 50-56 of SEQ ID NO:5, and CDR3 comprises a sequence set forth between amino acids 89-97 of SEQ ID NO: 5); or alternatively
(iii) V comprising CDR1, CDR2 and CDR3 H (wherein CDR1 comprises the sequence set forth between amino acids 31-35 of SEQ ID NO:37, CDR2 comprises the sequence set forth between amino acids 50-66 of SEQ ID NO:37, and CDR3 comprises the sequence set forth between amino acids 99-107 of SEQ ID NO: 37) or comprises the sequence set forth in SEQ ID NO:37, V of the sequence set forth in seq id no H V comprising CDR1, CDR2 and CDR3 L (wherein CDR1 comprises the sequence set forth between amino acids 24-34 of SEQ ID NO:5, CDR2 comprises the sequence set forth between amino acids 50-56 of SEQ ID NO:5, and CDR3 comprises a sequence that is at least about 66% identical (or 70% identical or 80% identical or 90% identical or 95% identical or 98% identical or 99% identical or 100% identical) to the sequence set forth between amino acids 89-97 of SEQ ID NO: 5).
In one embodiment, V of the protein L V containing antibody 8E2 L CDR1, CDR2 and CDR3 of (a) and V of the protein H V containing antibody 8E2 H CDR1 and CDR2 of (a).
In one embodiment, V of the protein L V containing antibody 8E2 L CDR1, CDR2 and CDR3 of (a) and V of the protein H V containing antibody 8E2 H CDR1 and CDR3 of (a).
In one embodiment, V of the protein L V containing antibody 8E2 L CDR1, CDR2 and CDR3 of (a) and V of the protein H V containing antibody 8E2 H CDR2 and CDR3 of (a).
In one embodiment, V of the protein L V containing antibody 8E2 L CDR1 and CDR3 of (a) and V of the protein H V containing antibody 8E2 H CDR1, CDR2, and CDR3 of (a).
In one embodiment, the antigen binding domain comprises:
(i) Comprising SEQ ID NO: 71V of the sequence set forth in SEQ ID NO. 71 H And a polypeptide comprising SEQ ID NO:35, V of the sequence set forth in SEQ ID NO. 35 L The method comprises the steps of carrying out a first treatment on the surface of the Or (b)
(ii) Comprising SEQ ID NO: 72V of the sequence set forth in SEQ ID NO. 72 H And a polypeptide comprising SEQ ID NO: 36V of the sequence set forth in SEQ ID NO. 36 L 。
Such IL-11 ra binding proteins may exhibit any one or more of the functional activities described herein, e.g., relative to SEQ ID NOs: 3, preferably binding to SEQ ID NO:3 polypeptide.
In one embodiment, the difference between the recited sequences and the IL-11Rα binding protein is a substitution.
One skilled in the art can determine the mutation site of the IL-11Rα binding proteins of the invention, e.g., within the framework region of a variable region-containing protein. In addition, the inventors identified V H CDR1, CDR2 and/or CDR3 and V L Multiple sites in CDR1 and/or CDR3 that can be mutated, multiple mutations that maintain or improve the activity of the IL-11 ra binding proteins of the invention. For example, the mutation (e.g., substitution) is within one or more (e.g., 2 or 3 or 4) of HCDR1 and/or HCDR2 of antibody 8E2 and/or one or more (e.g., 2 or 3 or 4 or 5 or 6) of the six N-terminal or six C-terminal residues of HCDR 3. For example, the mutation (e.g., substitution) is located within at least one (e.g., 2 or 3 or 4 or 5 or 6) of the six C-terminal amino acids of LCDR1 and/or one or more (e.g., 2 or 3 or 4 or 5) of the five N-terminal amino acids of LCDR3 of antibody 8E 2.
In one embodiment, the light chain CDR1 of the IL-11Rα binding protein of the invention comprises the following sequence:
QASQDX 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO：77)
wherein X is 1 =i or V; x is X 2 =n or D or G or S or a or H; x is X 3 =n or Y or I or K or M or Q or G or H; x is X 4 =y or W; x is X 5 =l or V or I orM and X 6 =n or E.
In one embodiment, the light chain CDR3 of the IL-11Rα binding proteins of the invention comprise the following sequence:
X 1 QX 2 X 3 X 4 X 5 X 6 PX 7 (SEQ ID NO：78)
wherein X is 1 =q or E or S or T; x is X 2 =y or H or F or N or S or W; x is X 3 =d or E; x is X 4 = N, D, F, S, E or T; x is X 5 =l or Q; x is X 6 =s or a or W or T or M or Q and X 7 =t or E or F or a or L or F or N or Q.
In one embodiment, X 1 Is Q and X 2 Is Y.
In one embodiment, X 6 Is S and X 7 Is T.
In one embodiment, the heavy chain CDR1 of the IL-11Rα binding protein of the invention comprises the following sequence:
X 1 X 2 SX 3 X 4
wherein X is 1 =w or R; x is X 2 =y or W or F; x is X 4 =m or I or V or T or L and X 5 =t or a
In one embodiment, the heavy chain CDR2 of the IL-11Rα binding protein of the invention comprises the following sequence:
SIVPX 1 X 2 X 3 X 4 TQYADSVKG
wherein X is 1 =s or W or Y or H; x is X 2 =g or a; x is X 3 =g or D or T and X 4 =h or Y or L or I or F
In one embodiment, the heavy chain CDR3 of the IL-11Rα binding protein of the invention comprises the following sequence:
X 1 X 2 X 3 WGX 4 FX 5 X 6
wherein X is 1 =g or P; x is X 2 =p or E or V or L or N or H; x is X 3 =g or D; x is X 4 =s or M or R or L; x is X 5 =d or a or W and X 6 =l or V or F or Q or E or Y or T
In one embodiment, X 1 Is G, X 2 Is P and X 3 Is G.
In one embodiment, X 5 Is D and X 6 Is L.
In one embodiment, the IL-11Rα binding protein comprises one of the foregoing consensus sequences as one CDR and the remaining CDRs are from antibody 8E2.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37-70, 74 or 76 and/or V of the antigen binding domain L Comprising SEQ ID NO:5-34, 73 or 75.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and comprises:
(i) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(ii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(iii) Comprising SEQ ID NO:38, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 38 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(iv) Comprising SEQ ID NO: 38V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(v) Comprising SEQ ID NO: 39V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 39 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(vi) Comprising SEQ ID NO:39, a sequence set forth in seq id noV of (2) H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vii) Comprising SEQ ID NO: 40V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 40 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(viii) Comprising SEQ ID NO:40, V of the sequence set forth in SEQ ID NO. 40 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ix) Comprising SEQ ID NO:41, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 41 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(x) Comprising SEQ ID NO: 41V of the sequence set forth in SEQ ID NO. 41 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xi) Comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 42 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xii) Comprising SEQ ID NO: 42V of the sequence set forth in SEQ ID NO. 42 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xiii) Comprising SEQ ID NO: 43V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 43 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xiv) Comprising SEQ ID NO: 43V of the sequence set forth in SEQ ID NO. 43 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xv) Comprising SEQ ID NO: 44V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 44 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xvi) Comprising SEQ ID NO: 44V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xvii) Comprising SEQ ID NO: 45V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 45 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xviii) Comprising SEQ ID NO: 45V of the sequence set forth in SEQ ID NO. 45 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xix) Comprising SEQ ID NO: 46V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 46 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xx) Comprising SEQ ID NO: 46V of the sequence set forth in SEQ ID NO. 46 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxi) Comprising SEQ ID NO: 47V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 47 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxii) Comprising SEQ ID NO:47, V of the sequence set forth in SEQ ID NO. 47 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxiii) Comprising SEQ ID NO: 48V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 48 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxiv) Comprising SEQ ID NO: 48V of the sequence set forth in SEQ ID NO. 48 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxv) Comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 49 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxvi) Comprising SEQ ID NO: 49V of the sequence set forth in SEQ ID NO. 49 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxvii) Comprising SEQ ID NO: 50V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 50 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxviii) Comprising SEQ ID NO: 50V of the sequence set forth in SEQ ID NO. 50 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxix) Comprising SEQ ID NO: 51V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 51 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxx) Comprising SEQ ID NO: 51V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxi) Comprising SEQ ID NO: 52V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 52 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxxii) Comprising SEQ ID NO: 52V of the sequence set forth in SEQ ID NO. 52 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxiii) Comprising SEQ ID NO: 53V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 53 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxxiv) Comprising SEQ ID NO: 53V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxv) Comprising SEQ ID NO: 54V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 54 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxxvi) Comprising SEQ ID NO: 54V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxvii) Comprising SEQ ID NO: 55V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 55 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xxxviii) Comprising SEQ ID NO: 55V of the sequence set forth in SEQ ID NO. 55 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxix) Bag(s)Comprising SEQ ID NO: 56V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 56 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xl) Comprising SEQ ID NO: 56V of the sequence set forth in SEQ ID NO. 56 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xli) Comprising SEQ ID NO: 57V of CDRs 1, 2 and 3 of the sequence set forth in column 57 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xlii) Comprising SEQ ID NO: 57V of the sequence set forth in SEQ ID NO. 57 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xliii) Comprising SEQ ID NO: 58V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 58 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xliv) Comprising SEQ ID NO: 58V of the sequence set forth in SEQ ID NO. 58 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xlv) Comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence listed in 59 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xlvi) Comprising SEQ ID NO: 59V of the sequence set forth in SEQ ID NO. 59 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xlvii) Comprising SEQ ID NO: 60V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 60 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(xlviii) Comprising SEQ ID NO: 60V of the sequence set forth in SEQ ID NO. 60 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xlix) Comprising SEQ ID NO:61, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 61 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:5, CDR1 of the sequence set forth in SEQ ID NO. 5,V of 2 and 3 L ；
(1) Comprising SEQ ID NO: 61V of the sequence set forth in SEQ ID NO. 61 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(li) comprises SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 62 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lii) comprising SEQ ID NO: 62V of the sequence set forth in SEQ ID NO. 62 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(liii) comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 63 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(liv) comprising SEQ ID NO: 63V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lv) comprises the amino acid sequence of SEQ ID NO: 64V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 64 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(1 vi) a polypeptide comprising SEQ ID NO: 64V of the sequence set forth in SEQ ID NO. 64 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vii) a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 65 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lviii) a polypeptide comprising SEQ ID NO: 65V of the sequence set forth in SEQ ID NO. 65 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lix) comprises SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 66 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lx) comprises SEQ ID NO: 66V of the sequence set forth in SEQ ID NO. 66 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxi) comprises the amino acid sequence of SEQ ID NO: v of CDRs 1, 2 and 3 of the 67-listed sequence H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lxii) comprising SEQ ID NO: 67V of the sequence set forth in SEQ ID NO. 67 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxiii) comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 68 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lxiv) comprising SEQ ID NO: 68V of the sequence set forth in SEQ ID NO. 68 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxv) comprises the amino acid sequence of SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence listed in 69 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lxvi) a polypeptide comprising SEQ ID NO: 69V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxvii) a polypeptide comprising SEQ ID NO: 70V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 70 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lxviii) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxix) comprises SEQ ID NO: 70V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 70 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(lxx) comprises the amino acid sequence of SEQ ID NO:70, V of the sequence set forth in SEQ ID NO. 70 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(lxxi) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally, further comprising an amino acid in the N-terminal direction of CDR 1) and a bagComprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 6 L ；
(lxxxii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 6V of the sequence set forth in SEQ ID NO. 6 L ；
(lxxxiii) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in Table 7 L ；
(lxxiv) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 7V of the sequence set forth in L ；
(lxxxv) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 8 L ；
(lxxvi) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 8V of the sequence set forth in SEQ ID NO. 8 L ；
(lxxxvii) a polypeptide comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 9 L ；
(lxxviii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 9V of the sequence set forth in SEQ ID NO. 9 L ；
(lxxix) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 10 L ；
(lxxx) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 10V of the sequence set forth in seq id no L ；
(lxxxi) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:11, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 11 L ；
(lxxii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 11V of the sequence set forth in seq id no L ；
(lxxiii) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:12, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 12 L ；
(lxxiv) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 12V of the sequence set forth in SEQ ID NO. 12 L ；
(lxxxv) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 13V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 13 L ；
(lxxxvi) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 13V of the sequence set forth in SEQ ID NO. 13 L ；
(lxxxvii) comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:14, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 14 L ；
(lxxxviii) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 14V of the sequence set forth in seq id no L ；
(lxxix) comprises SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:15, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 15 L ；
(xc) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 15V of the sequence set forth in seq id no L ；
(xci) comprises SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:16, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 16 L ；
(xcii) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 16V of the sequence set forth in seq id no L ；
(xciii) comprises SEQ ID NO:37, and a CDR of the sequence set forth in seq id no1. V of 2 and 3 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:17, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 17 L ；
(xciv) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 17V of the sequence set forth in SEQ ID NO. 17 L ；
(xcv) comprises the amino acid sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:18, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 18 L ；
(xcvi) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 18V of the sequence set forth in SEQ ID NO. 18 L ；
(xcvii) a polypeptide comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:19, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 19 L ；
(xcviii) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 19V of the sequence set forth in SEQ ID NO. 19 L ；
(xcix) comprises SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:20, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 20 L ；
(c) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 20V of the sequence set forth in seq id no L ；
(ci) comprises SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 21 L ；
(cii) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:21, V of the sequence set forth in SEQ ID NO. 21 L ；
(ciii) comprises the amino acid sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 22V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 22 L ；
(civ) comprises the amino acid sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 22V of the sequence set forth in SEQ ID NO. 22 L ；
(cv) comprises the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:23, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 23 L ；
(cvi) comprises the amino acid sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:23, V of the sequence set forth in SEQ ID NO. 23 L ；
(vii) a polypeptide comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:24, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 24 L ；
(cvviii) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:24, V of the sequence set forth in SEQ ID NO. 24 L ；
(cix) comprises SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:25, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 25 L ；
(cx) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:25, V of the sequence set forth in SEQ ID NO. 25 L ；
(cxi) comprising the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:26, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 26 L ；
(cxii) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:26, V of the sequence set forth in SEQ ID NO. 26 L ；
(cxiii) comprising the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:27, CDRs 1, 2 and 3V of the sequence set forth in seq id no L ；
(cxiv) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 27V of the sequence set forth in seq id no L ；
(cxv) comprises the amino acid sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 28V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 28 L ；
(cxvi) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 28V of the sequence set forth in seq id no L ；
(cxvii) a polypeptide comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 29V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 29 L ；
(cxviii) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 29V of the sequence set forth in seq id no L ；
(cxix) comprising the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:30, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 30 L ；
(cxx) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 30V of the sequence set forth in SEQ ID NO. 30 L ；
(cxxi) comprising the sequence set forth in SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 31V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 31 L ；
(cxxxii) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 31V of the sequence set forth in seq id no L ；
(cxxxiii) comprising the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 32V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 32 L ；
(cxxxiv) comprising the sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 32V of the sequence set forth in seq id no L ；
(cxxxv) comprising the sequence of SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally, further comprising an amino acid in the direction of the N-terminus of CDR 1) andcomprising SEQ ID NO:33, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 33 L ；
(cxxxvi) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 33V of the sequence set forth in seq id no L ；
(cxxxvii) a polypeptide comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:34, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 34 L The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively
(cxxxviii) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:34, V of the sequence set forth in seq id no L 。
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and comprises:
(i) Comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 49 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(ii) Comprising SEQ ID NO: 49V of the sequence set forth in SEQ ID NO. 49 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(iii) Comprising SEQ ID NO: 53V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 53 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(iv) Comprising SEQ ID NO: 53V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(v) Comprising SEQ ID NO: 57V of CDRs 1, 2 and 3 of the sequence set forth in column 57 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(vi) Comprising SEQ ID NO: 57V of the sequence set forth in SEQ ID NO. 57 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vii) Comprising SEQ ID NO:58 CDR1, 2 of the sequence set forth in seq id noAnd V of 3 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(viii) Comprising SEQ ID NO: 58V of the sequence set forth in SEQ ID NO. 58 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ix) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 8 L ；
(x) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 8V of the sequence set forth in SEQ ID NO. 8 L ；
(xi) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:15, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 15 L ；
(xii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 15V of the sequence set forth in seq id no L ；
(xiii) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:16, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 16 L ；
(xiv) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 16V of the sequence set forth in seq id no L ；
(xv) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:18, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 18 L ；
(xvi) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 18V of the sequence set forth in SEQ ID NO. 18 L ；
(xvii) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 29V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 29 L The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively
(xviii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 29V of the sequence set forth in seq id no L 。
In one embodiment, these CDRs are as follows:
(i) Heavy chain CDR1: amino acids 31-35 of the recited sequence;
(ii) Heavy chain CDR2: amino acids 50-66 of the recited sequence (optionally, any one or more of the five C-terminal amino acids are substituted with another naturally occurring amino acid);
(iii) Heavy chain CDR3: amino acids 99-107 of the recited sequence;
(iv) Light chain CDR1: amino acids 24-34 of the recited sequence;
(v) Light chain CDR2: amino acids 50-56 of the recited sequence; and
(vi) Light chain CDR3: amino acids 89-97 of the recited sequences.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:49 and V of the antigen binding domain of CDR1, 2 and 3 of the sequence listed in SEQ ID NO. 49 L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:49 and V of the antigen binding domain L Comprising SEQ ID NO: 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:53 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:53 and V of the antigen binding domain L Comprising SEQ ID NO: 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:57 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:57 and V of the antigen binding domain of the antigen L Comprising SEQ ID NO: 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:58 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:58, V of the antigen binding domain L Comprising SEQ ID NO: 5.
The invention also provides a kit comprising an antibodyAn IL-11Rα binding protein of an antigen binding domain that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and V of the antigen binding domain H Comprising SEQ ID NO:37 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO:8, CDR1, 2 and 3 of the sequence listed in seq id no.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 and V of the antigen binding domain L Comprising SEQ ID NO: 8.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO:15, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 and V of the antigen binding domain L Comprising SEQ ID NO: 15.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO:16, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also providesAn IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11 ra and neutralizes IL-11 signaling and V of the antigen binding domain H Comprising SEQ ID NO:37 and V of the antigen binding domain L Comprising SEQ ID NO: 16.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO:18, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 and V of the antigen binding domain L Comprising SEQ ID NO: 18.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 (optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and V of the antigen binding domain L Comprising SEQ ID NO:29, CDR 1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain H Comprising SEQ ID NO:37 and V of the antigen binding domain L Comprising SEQ ID NO: 29.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody,the antigen binding domain binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and V of the antigen binding domain H Comprising SEQ ID NO: 37. In one embodiment, V L Comprising SEQ ID NO:18, CDR 1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an IL-11Rα binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11Rα and neutralizes IL-11 signaling and the V of the antigen binding domain L Comprising SEQ ID NO: 18. In one embodiment, V H Comprising SEQ ID NO:37, CDR 1, 2 and 3 of the sequence set forth in seq id no.
In one embodiment, these CDRs are as follows:
(i) Heavy chain CDR1: amino acids 31-35 of the recited sequence;
(ii) Heavy chain CDR2: amino acids 50-66 of the recited sequence (optionally, any one or more of the five C-terminal amino acids are substituted with another naturally occurring amino acid);
(iii) Heavy chain CDR3: amino acids 99-107 of the recited sequence;
(iv) Light chain CDR1: amino acids 24-34 of the recited sequence;
(v) Light chain CDR2: amino acids 50-56 of the recited sequence; and
(vi) Light chain CDR3: amino acids 89-97 of the recited sequences.
In one embodiment, the IL-11Rα binding protein described herein comprises at least one V H And a V L Wherein the V is H And V L Binding forms Fv comprising an antigen binding domain. It will be appreciated by those skilled in the art that the antigen binding domain comprises the binding site of an antibody.
In one embodiment, the V H And the V is L Is co-located in a single polypeptide chain. For example, the protein is:
(i) Single chain Fv fragments (scFv);
(ii) Dimeric scFv (di-scFv);
(iii) With the constant region Fc or heavy chain constant domain (C) H ) 2 and/orC H 3 to one of (i) or (ii); or alternatively
(iv) One of (i) or (ii) linked to a protein that binds immune effector cells.
In one embodiment, the V L And V H Are individual polypeptide chains.
For example, the protein is:
(i) A diabody;
(ii) A tri-antibody;
(iii) A four-antibody;
(iv)Fab；
(v)F(ab’) 2 ；
(vi、)Fv；
(vii) With the constant region Fc or heavy chain constant domain (C) H ) 2 and/or C H 3 to one of (i) to (vi);
(viii) One of (i) to (vi) linked to a protein that binds immune effector cells.
The foregoing proteins (described in the preceding two lists) may also be referred to as antigen binding domains of antibodies.
In one embodiment, the protein is an antibody, e.g., a monoclonal antibody. In one embodiment, the antibody is a naked antibody.
In one embodiment, the protein (or antibody) is chimeric, deimmunized, humanized, human or primatized.
In one embodiment, the protein or antibody is human.
Additionally or alternatively, the invention provides an antibody that binds to IL-11 ra and neutralizes IL-11 signaling, the antibody comprising:
(i) Comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 49 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(ii) Comprising SEQ ID NO: 49V of the sequence set forth in SEQ ID NO. 49 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(iii) Comprising SEQ ID NO: 53V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 53 H (any one of)Optionally further comprising an amino acid in the direction of the N-terminus of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(iv) Comprising SEQ ID NO: 53V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(v) Comprising SEQ ID NO: 57V of CDRs 1, 2 and 3 of the sequence set forth in column 57 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(vi) Comprising SEQ ID NO: 57V of the sequence set forth in SEQ ID NO. 57 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vii) Comprising SEQ ID NO: 58V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 58 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 5 L ；
(viii) Comprising SEQ ID NO: 58V of the sequence set forth in SEQ ID NO. 58 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ix) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: v of CDRs 1, 2 and 3 of the sequence set forth in 8 L ；
(x) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 8V of the sequence set forth in SEQ ID NO. 8 L ；
(xi) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:15, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 15 L ；
(xii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 15V of the sequence set forth in seq id no L ；
(xiii) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:16, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 16 L ；
(xiv) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 16V of the sequence set forth in seq id no L ；
(xv) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO:18, V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 18 L ；
(xvi) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 18V of the sequence set forth in SEQ ID NO. 18 L ；
(xvii) Comprising SEQ ID NO: 37V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 37 H (optionally further comprising an amino acid in the N-terminal direction of CDR 1) and a polypeptide comprising SEQ ID NO: 29V of CDRs 1, 2 and 3 of the sequence set forth in SEQ ID NO. 29 L The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively
(xviii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 29V of the sequence set forth in seq id no L 。
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO: 37. In one embodiment, V L Comprising SEQ ID NO:18, CDR1, 2 and 3 of the sequence set forth in seq id no.
(xix) The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody L Comprising SEQ ID NO: 18. In one embodiment, V H Comprising SEQ ID NO:37, CDR1, 2 and 3 of the sequence set forth in seq id no.
In one embodiment, these CDRs are as follows:
(i) Heavy chain CDR1: amino acids 31-35 of the recited sequence;
(ii) Heavy chain CDR2: amino acids 50-66 of the recited sequence (optionally, any one or more of the five C-terminal amino acids are substituted with another naturally occurring amino acid);
(iii) Heavy chain CDR3: amino acids 99-107 of the recited sequence;
(iv) Light chain CDR1: amino acids 24-34 of the recited sequence;
(v) Light chain CDR2: amino acids 50-56 of the recited sequence; and
(vi) Light chain CDR3: amino acids 89-97 of the recited sequences.
Exemplary V H And V L The sequences of (2) are described in Table 1.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:49 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:49 and V of the antibody L Comprising SEQ ID NO: 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:53 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:53 and V of the antibody L Comprising SEQ ID NO: 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:57 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:57 and V of the antibody L Comprising SEQ ID NO: 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:58 (optionally also containing CDRs in sequence1N terminal amino acid) and V of the antibody L Comprising SEQ ID NO: CDR1, 2 and 3 of the sequence listed in 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:58 and V of the antibody L Comprising SEQ ID NO: 5.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO:8, CDR1, 2 and 3 of the sequence listed in seq id no.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 and V of the antibody L Comprising SEQ ID NO: 8.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO:15, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 and V of the antibody L Comprising SEQ ID NO: 15.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO:16, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 and V of the antibody L Comprising SEQ ID NO: 16.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO:18, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 and V of the antibody L Comprising SEQ ID NO: 18.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 (optionally also containing an amino acid in the direction of the N-terminus of CDR 1) and V of the antibody L Comprising SEQ ID NO:29, CDR1, 2 and 3 of the sequence set forth in seq id no.
The invention also provides an antibody that binds IL-11Rα and neutralizes IL-11 signaling, V of the antibody H Comprising SEQ ID NO:37 and V of the antibody L Comprising SEQ ID NO: 29.
In one embodiment, these CDRs are as follows:
(i) Heavy chain CDR1: amino acids 31-35 of the recited sequence;
(ii) Heavy chain CDR2: amino acids 50-66 of the recited sequence (optionally, any one or more of the five C-terminal amino acids are substituted with another naturally occurring amino acid);
(iii) Heavy chain CDR3: amino acids 99-107 of the recited sequence;
(iv) Light chain CDR1: amino acids 24-34 of the recited sequence;
(v) Light chain CDR2: amino acids 50-56 of the recited sequence; and
(vi) Light chain CDR3: amino acids 89-97 of the recited sequences.
Reference herein to a protein or antibody that "binds" to IL-11R alpha provides literal support for a protein or antibody that "specifically binds" or "specifically binds" to IL-11R.
The invention also provides an antigen binding domain or antigen binding fragment of the aforementioned antibodies.
In one embodiment, the proteins or antibodies described herein comprise a human constant region, such as an IgG constant region, e.g., an IgG1, igG2, igG3, or IgG4 constant region, or a mixture thereof. For inclusion of V H And V L The antibody or protein of V H May be linked to the heavy chain constant region and the V L May be linked to the light chain constant region.
Whole antibodies of the invention (or comprising constant regions or C H 3 or an IL-11 ra binding protein) may be removed, for example during antibody or protein production or purification, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a whole antibody (or IL-11R binding protein) may comprise a population in which all C-terminal lysine residues have been removed, a population in which no C-terminal lysine residues have been removed, and/or a population comprising a mixture of proteins with and without C-terminal lysine residues. In some embodiments, the populations may further comprise proteins in which the C-terminal lysine residue in one of the heavy chain constant regions is removed. Similarly, the composition of whole antibodies may comprise the same or similar mixture of populations of antibodies with or without C-terminal lysine residues.
In one embodiment, the proteins or antibodies described herein comprise the constant region of an IgG4 antibody or the stabilized constant region of an IgG4 antibody. In one embodiment, the protein or antibody comprises an IgG4 constant region with proline at position 241 (according to the Kabat coding system (Kabat et al, sequences of Proteins of Immunological Interest (protein sequence of immunological interest), washington, d.c., department of health and human services, 1987 and/or 1991)).
In one embodiment, the heavy chain constant region comprises SEQ ID NO:83 from bit 119 to bit 445. In one embodiment, a protein or antibody described herein or a composition of a protein or antibody described herein comprises a heavy chain constant region, including a stabilized heavy chain constant region, including a mixture of sequences with or without all or part of a C-terminal lysine residue.
In one embodimentAntibodies of the invention comprise a V described herein linked to or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as described above) H And V is L Linked to or fused to a kappa light chain constant region.
The functional properties of the IL-11Rα binding proteins of the invention will be mutatis mutandis for use in the antibodies of the invention.
In one embodiment, the IL-11Rα binding proteins or antibodies described herein are isolated and/or recombinant.
In one embodiment, the IL-11Rα binding proteins or antibodies of the invention are conjugated to another compound, such as a detectable label or a compound that extends the half-life of the protein or antibody, such as polyethylene glycol or albumin binding protein. Other suitable compounds are described herein.
The invention also provides a nucleic acid encoding an IL-11Rα binding protein or antibody or polypeptide thereof of the invention.
In one embodiment, such nucleic acid is contained in an expression construct, wherein the nucleic acid is operably linked to a promoter. Such expression constructs may be in a vector (e.g., a plasmid).
In embodiments of the invention involving a single polypeptide chain IL-11Rα binding protein, the expression construct may comprise a promoter linked to the nucleic acid encoding the polypeptide chain.
In embodiments of the invention involving multiple polypeptide chain IL-11Rα binding proteins, the expression construct comprises a polypeptide encoding a polypeptide comprising, for example, V operably linked to a promoter H Is encoded by a polypeptide comprising, for example, V operably linked to a promoter L Is a polypeptide of (a).
In another embodiment, the expression construct is a bicistronic expression construct, e.g., comprising the following components operably linked in 5 'to 3' order:
(i) Promoters
(ii) A nucleic acid encoding a first polypeptide;
(iii) An internal ribosome entry site; and
(iv) A nucleic acid encoding a second polypeptide,
wherein the first polypeptide comprises V H And the second polypeptide comprises V L And vice versa.
The invention also contemplates independent expression constructs, one of which encodes a polypeptide comprising V H And the other code comprises V L Is a second polypeptide of (a). For example, the present invention also provides a composition comprising:
(i) A first expression construct comprising a vector encoding a vector comprising V operably linked to a promoter H Is a polypeptide of (a); and
(ii) A second expression construct comprising a vector encoding a vector comprising V operably linked to a promoter L Is a polypeptide of (a).
The invention also provides an isolated or recombinant cell that expresses an IL-11Rα binding protein of the invention.
In one embodiment, the cell comprises an expression construct of the invention or:
(i) A first expression construct comprising a vector encoding a vector comprising V operably linked to a promoter H Is a polypeptide of (a); and
(ii) A second expression construct comprising a vector encoding a vector comprising V operably linked to a promoter L Is a nucleic acid of a polypeptide of (a),
wherein the first and second polypeptides are combined to form an IL-11Rα binding protein of the invention.
Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.
The invention also provides methods of producing the IL-11Rα binding proteins or antibodies of the invention. For example, such methods involve maintaining the expression constructs of the invention under conditions sufficient to produce an IL-11Rα binding protein or antibody.
In one embodiment, the method of producing an IL-11Rα binding protein or antibody of the invention comprises culturing a cell of the invention under conditions sufficient to produce and optionally secrete an IL-11R d binding protein or antibody.
In one embodiment, the method of producing an IL-11Rα binding protein or antibody of the invention further comprises isolating the protein or antibody and optionally formulating the IL-11Rα binding protein or antibody as a pharmaceutical composition.
The invention also provides a composition comprising an IL-11 ra binding protein or antibody of the invention and a pharmaceutically acceptable carrier.
In some embodiments, the composition comprises:
(i) An antibody of the invention comprising a C-terminal lysine residue from a heavy chain;
(ii) Antibodies of the invention lacking a C-terminal lysine residue from the heavy chain; and/or
(iii) An antibody of the invention comprising a C-terminal lysine residue on one heavy chain and lacking a C-terminal lysine residue on the other (or other) heavy chain,
And (optionally) a pharmaceutically acceptable carrier.
The invention also provides a method of treating or preventing an IL-11 mediated condition in a subject comprising administering an IL-11Rα binding protein, antibody or composition of the invention. In this aspect, the IL-11Rα binding protein, antibody or composition can be used to prevent the recurrence of a disorder, and this is considered to be preventing the disorder.
In one embodiment, the IL-11 mediated disorder may be an autoimmune disorder, an inflammatory disorder, a wasting disorder, a bone disorder, or cancer.
Exemplary autoimmune disorders include arthritis, inflammatory bowel disease, and psoriasis.
Exemplary inflammatory conditions include infection-induced inflammation (e.g., mycobacterium tuberculosis-induced inflammation), gastritis (e.g., associated with gastric cancer), inflammatory tracheal disorders (e.g., asthma, chronic Obstructive Pulmonary Disease (COPD), rhinitis, or allergy), or inflammatory dermatitis (e.g., atopic dermatitis).
Exemplary wasting conditions include cachexia (e.g., cachexia resulting from cancer cachexia or renal failure) or sarcopenia.
Exemplary bone disorders include bone resorption/damage caused by osteoporosis (including postmenopausal osteoporosis), bone fractures, cancer (e.g., metastatic bone cancer, myeloma, or paget's disease), and bone resorption/damage caused by cancer therapy (e.g., chemotherapy, hormone removal, or hormone inhibition).
Exemplary cancers include hematologic cancers, cancers of epithelial origin, liver cancer, pancreatic cancer, gastric cancer, osteosarcoma, endometrial cancer, and ovarian cancer.
In one embodiment, the cancer or bone disorder is metastasis of a cancer to a bone.
The invention also provides a method of inhibiting or neutralizing IL-11 in a subject comprising administering an IL-11Rα binding protein, antibody or composition of the invention. In one embodiment, the subject has an IL-11 mediated condition.
The invention also provides a method of preventing pregnancy in a subject comprising administering an IL-11Rα binding protein, antibody or composition of the invention.
In one embodiment, the methods described herein comprise administering from about 0.05mg/kg to about 30mg/kg of an IL-11Rα binding protein or antibody. For example, the method comprises administering from about 0.1mg/kg to about 10mg/kg or from about 0.2mg/kg to about 5mg/kg of IL-11Rα binding protein or antibody. In one embodiment, the method comprises administering about 0.5-2.0mg/kg of IL-11Rα binding protein or antibody.
The invention also provides the use of an IL-11 ra binding protein or antibody as described in any of the examples in medicine.
The invention also provides the use of an IL-11Rα binding protein or antibody as described in any of the examples in the manufacture of a medicament for the treatment of an IL-11 mediated disorder. Exemplary disorders are described herein.
The invention also provides a method of locating and/or detecting and/or diagnosing and/or predicting an IL-11 mediated disorder associated with a cell expressing IL-11Rα, the method comprising detecting in vivo an IL-11Rα binding protein or antibody described herein (if present) that binds to a cell expressing IL-11Rα, wherein the IL-11Rα binding protein or antibody is coupled to a detectable label.
In one embodiment, the method further comprises administering the IL-11Rα binding protein to a subject.
The invention also provides a method of detecting IL-11Rα or a cell expressing IL-11Rα in a sample, the method comprising contacting the sample with a protein or antibody as described in any of the examples to form a complex and detecting the complex, wherein detection of the complex indicates the presence of IL-11Rα or a cell expressing IL-1Rα in the sample. In one embodiment, the method is performed ex vivo or in vitro.
The invention also provides a method of diagnosing or prognosticating an IL-11 mediated disorder, the method comprising performing the method of any of the embodiments to detect IL-11Rα or cells expressing IL-11Rα, wherein detection of IL-11Rα or cells expressing IL-1Rα is diagnostic or predictive of the disorder. In one embodiment, the method is performed ex vivo or in vitro. Exemplary IL-11Rα mediated disorders are described herein.
The invention also provides a kit (e.g., package or article of manufacture) comprising an IL-11 ra binding protein or antibody as described in any of the embodiments, optionally together with instructions for use in the methods described herein.
Key points of the sequence table
SEQ ID NO 1: amino acid sequence of Homo sapiens IL-11Rα precursor
SEQ ID NO 2: amino acid sequence of cynomolgus monkey (Macaca fascicularis) IL-11Rα precursor
SEQ ID NO 3: comprising SEQ ID NO:1, an 8xHIS tag and a nucleotide sequence corresponding to SEQ ID NO:1 (also referred to as "WT F/L") of serine at position 248 of the amino acid sequence
SEQ ID NO 4: amino acid sequence of homo sapiens gp130
SEQ ID NO 5: v of antibody 8E2 L Amino acid sequence of chain
SEQ ID NO 6: v of antibody TS-303 L Amino acid sequence of chain
SEQ ID NO 7: v of antibody TS-305 L Amino acid sequence of chain
SEQ ID NO 8: v of antibody TS-306 L Amino acid sequence of chain
SEQ ID NO 9: v of antibody TS-307 L Amino acid sequence of chain
SEQ ID NO 10: v of antibody TS-310 L Amino acid sequence of chain
SEQ ID NO 11: v of antibody TS-311 L Amino acid sequence of chain
SEQ ID NO 12: v of antibody TS-312 L Amino acid sequence of chain
SEQ ID NO 13: v of antibody TS-313 L Amino acid sequence of chain
SEQ ID NO 14: v of antibody TS-322 L Amino acid sequence of chain
SEQ ID NO 15: v of antibody TS-2 L Amino acid sequence of chain
SEQ ID NO 16: v of antibody TS-4 L Amino acid sequence of chain
SEQ ID NO 17: v of antibody TS-6 L Amino acid sequence of chain
SEQ ID NO 18: v of antibody TS-7 L Amino acid sequence of chain
SEQ ID NO 19: v of antibody TS-9 L Amino acid sequence of chain
SEQ ID NO 20: v of antibody TS-13 L Amino acid sequence of chain
SEQ ID NO 21: v of antibody TS-14 L Amino acid sequence of chain
SEQ ID NO 22: v of antibody TS-17 L Amino acid sequence of chain
SEQ ID NO 23: v of antibody TS-20 L Amino acid sequence of chain
SEQ ID NO 24: v of antibody TS-21 L Amino acid sequence of chain
SEQ ID NO 25: v of antibody TS-22 L Amino acid sequence of chain
SEQ ID NO 26: v of antibody TS-29 L Amino acid sequence of chain
SEQ ID NO 27: v of antibody TS-32 L Amino acid sequence of chain
SEQ ID NO 28: v of antibody TS-49 L Amino acid sequence of chain
SEQ ID NO 29: v of antibody TS-51 L Amino acid sequence of chain
SEQ ID NO 30: v of antibody TS-55 L Amino acid sequence of chain
SEQ ID NO31: v of antibody TS-57 L Amino acid sequence of chain
SEQ ID NO 32: v of antibody TS-58 L Amino acid sequence of chain
SEQ ID NO 33: v of antibody TS-63 L Amino acid sequence of chain
SEQ ID NO 34: v of antibody TS-64 L Amino acid sequence of chain
SEQ ID NO 35: v of 8E2 antibodies and derivatives L Consensus amino acid sequence of the chain
SEQ ID NO 36: v of 8E2 antibodies and selected derivatives L Consensus amino acid sequence of the chain
SEQ ID NO 37: v of antibody 8E2 H Amino acid sequence of chain
SEQ ID NO 38: v of antibody TS-66 H Amino acid sequence of chain
SEQ ID NO 39: v of antibody TS-69 H Amino acid sequence of chain
SEQ ID NO 40: v of antibody TS-71 H Amino acid sequence of chain
SEQ ID NO 41: v of antibody TS-76 H Amino acid sequence of chain
SEQ ID NO 42: v of antibody TS-79 H Amino acid sequence of chain
SEQ ID NO 43: v of antibody TS-82 H Amino acid sequence of chain
SEQ ID NO 44: v of antibody TS-88 H Amino acid sequence of chain
SEQ ID NO 45: v of antibody TS-89 H Amino acid sequence of chain
SEQ ID NO 46: v of antibody TS-91 H Amino acid sequence of chain
SEQ ID NO 47: v of antibody TS-92 H Amino acid sequence of chain
SEQ ID NO 48: v of antibody TS-97 H Amino acid sequence of chain
SEQ ID NO 49: v of antibody TS-101 H Amino acid sequence of chain
SEQ ID NO 50: v of antibody TS-103 H Amino acid sequence of chain
SEQ ID NO51: v of antibody TS-104 H Amino acid sequence of chain
SEQ ID NO 52: v of antibody TS-107 H Amino acid sequence of chain
SEQ ID NO 53: v of antibody TS-108 H Amino acid sequence of chain
SEQ ID NO 54: v of antibody TS-115 H Amino acid sequence of chain
SEQ ID NO 55: v of antibody TS-129 H Amino acid sequence of chain
SEQ ID NO 56: v of antibody TS-133 H Amino acid sequence of chain
SEQ ID NO 57: v of antibody TS-134 H Amino acid sequence of chain
SEQ ID NO 58: v of antibody TS-135 H Amino acid sequence of chain
SEQ ID NO 59: v of antibody TS-136 H Amino acid sequence of chain
SEQ ID NO 60: v of antibody TS-140 H Amino acid sequence of chain
SEQ ID NO 61: v of antibody TS-143 H Amino acid sequence of chain
SEQ ID NO 62: v of antibody TS-151 H Amino acid sequence of chain
SEQ ID NO 63: v of antibody TS-156 H Amino acid sequence of chain
SEQ ID NO 64: v of antibody TS-213 H Amino acid sequence of chain
SEQ ID NO 65: v of antibody TS-214 H Amino acid sequence of chain
SEQ ID NO 66: v of antibody TS-215 H Amino acid sequence of chain
SEQ ID NO 67: v of antibody TS-218 H Amino acid sequence of chain
SEQ ID NO 68: v of antibody TS-221 H Amino acid sequence of chain
SEQ ID NO 69: v of antibody TS-222 H Amino acid sequence of chain
SEQ ID NO 70: v of antibody TS-224 H Amino acid sequence of chain
SEQ ID NO 71: v of 8E2 antibodies and derivatives H Consensus amino acid sequence of the chain
SEQ ID NO 72: v of 8E2 antibodies and selected derivatives H Consensus amino acid sequence of the chain
SEQ ID NO 73: v of antibody 8E4 L Amino acid sequence of chain
SEQ ID NO 74: v of antibody 8E4 H Amino acid sequence of chain
SEQ ID NO 75: v of antibody 8D10 L Amino acid sequence of chain
SEQ ID NO 76: v of antibody 8D10 H Amino acid sequence of chain
SEQ ID NO 77: v of 8E2 antibodies and derivatives L Consensus amino acid sequence of CDR1 of the chain
SEQ ID NO 78: v of 8E2 antibodies and derivatives L Consensus amino acid sequence of CDR3 of chain
SEQ ID NO 79: v of 8E2 antibodies and derivatives H Consensus amino acid sequence of CDR1 of the chain
SEQ ID NO 80: v of 8E2 antibodies and derivatives H Consensus amino acid sequence of CDR2 of the chain
SEQ ID NO 81: v of 8E2 antibodies and derivatives H Consensus amino acid sequence of CDR3 of chain
SEQ ID NO 82: amino acid sequence of mouse (Mus musculus) IL-11Rα precursor
SEQ ID NO 83: amino acid sequence of heavy chain of antibody 8E2
SEQ ID NO 84: amino acid sequence of light chain of antibody 8E2
SEQ ID NO 85: amino acids 23-318, 8xHIS tag comprising homo sapiens IL-11Rα (SEQ ID NO: 1) and located corresponding to SEQ ID NO:1 (also referred to as "WT D1/2") of serine at position 248 of the amino acid sequence
SEQ ID NO:86: amino acid sequence of polypeptide comprising amino acids 23-110 of homo sapiens IL-11Rα (SEQ ID NO: 1) and amino acids 111-367 of mouse IL-11Rα (SEQ ID NO: 82) wherein serine is present at position 206 corresponding to SEQ ID NO:82 and 8xHIS tag
SEQ ID NO:87: amino acid sequence of polypeptide comprising amino acids 23-215 of homo sapiens IL-11Rα (SEQ ID NO: 1) and amino acids 216-367 of mouse IL-11Rα (SEQ ID NO: 82) and 8xHIS tag
SEQ ID NO:88: amino acid sequence of polypeptide comprising amino acids 23-318 of homo sapiens IL-11Rα (SEQ ID NO: 1) wherein serine is present at the position corresponding to position 248 of SEQ ID NO:1 and amino acids 319-367 of mouse IL-11Rα (SEQ ID NO: 82) and 8xHIS tag
SEQ ID NO:89: amino acid sequence of polypeptide comprising amino acids 24-215 of mouse IL-11Rα (SEQ ID NO: 82) wherein serine is present at position 206 corresponding to SEQ ID NO:82 and amino acids 216-363 of homo sapiens IL-11Rα (SEQ ID NO: 1) and 8xHIS tag
SEQ ID NO 90: amino acids 24-318, 8xHIS tag comprising mouse sIL-11Rα (SEQ ID NO: 82) and located at a position corresponding to SEQ ID NO:82, serine at position 206, and a polypeptide amino acid sequence of serine at position 206
SEQ ID NO 91: amino acid sequence of light chain of antibody 8E4
SEQ ID NO 92: amino acid sequence of heavy chain of antibody 8E4
SEQ ID NO 93: amino acid sequence of light chain of antibody 8D10
SEQ ID NO 94: amino acid sequence of heavy chain of antibody 8D10
SEQ ID NO:95: in the sequence corresponding to SEQ ID NO:1, comprising glutamic acid at position 117 of SEQ ID NO:85 amino acid sequence
SEQ ID NO:96: in the sequence corresponding to SEQ ID NO:1, comprising arginine at position 66 of SEQ ID NO:85 amino acid sequence
SEQ ID NO:97: in the sequence corresponding to SEQ ID NO:1, a serine-containing SEQ ID NO:85 amino acid sequence
SEQ ID NO:98: in the sequence corresponding to SEQ ID NO:1, a serine-containing SEQ ID NO:85 amino acid sequence
SEQ ID NO:99: in the sequence corresponding to SEQ ID NO:1, comprising alanine at position 178 of SEQ ID NO:85 amino acid sequence
SEQ ID NO:100: amino acid sequence of mature homo sapiens IL-11Rα
SEQ ID NO:101: amino acid sequence of mature cynomolgus monkey IL-11Rα
SEQ ID NO:102: amino acid sequence of mature mouse sIL-11Rα
SEQ ID NO:103: the consensus amino acid sequence of 8E2L1 shown in FIG. 1
SEQ ID NO:104: the consensus amino acid sequence of 8E2L3.1 shown in FIG. 1
SEQ ID NO:105: the consensus amino acid sequence of 8E2L3.2 shown in FIG. 1
SEQ ID NO:106: FIG. 2 shows the consensus amino acid sequence of 8E2H1
SEQ ID NO:107: FIG. 2 shows the consensus amino acid sequence of 8E2H2
SEQ ID NO:108: the consensus amino acid sequence of 8E2H3.1 shown in FIG. 2
SEQ ID NO:109: the consensus amino acid sequence of 8E2H3.2 shown in FIG. 2
SEQ ID NO:110: amino acid sequence of antagonistic IL-11 mutein
Drawings
FIG. 1 shows antibody 8E2 and its affinity maturation form V L Is a single CDR. The consensus sequences listed (SEQ ID NOS: 103-105) represent the most frequently occurring amino acids at each position and were determined using the MegAlign software. The sequences derived from the antibodies are listed in Table 1, except for the consensus sequences.
FIG. 2 shows antibody 8E2 and its affinity maturation form V H Is a single CDR. The consensus sequences listed (SEQ ID NOS: 106-109) represent the most frequently occurring amino acids at each position and were determined using the MegAlign software. The sequences derived from the antibodies are listed in Table 1, except for the consensus sequences.
FIGS. 3A, B, C and D show the sequences of the variable regions of 8E2 antibodies and derivatives. FIG. 3A shows V of 8E2 and its antibody derivatives L Sequence of region and V of 8E2 and antibody derivatives thereof L Consensus sequence of the region. FIG. 3B shows V of 8E2 and selected antibody derivatives L Sequence of region and V of 8E2 and selected antibody derivatives L Consensus sequence of the region. FIG. 3C shows V of 8E2 and its antibody derivatives H Sequence of region and V of 8E2 and antibody derivatives thereof H Consensus sequence of the region. Drawing of the figure3D shows V of 8E2 and selected antibody derivatives H Sequence of region and V of 8E2 and selected antibody derivatives H Consensus sequence of the region. The boxed region contains the CDRs defined by the Kabat coding system (as shown).
FIG. 4 includes a series of schematic diagrams showing that IL-11 stimulates phosphorylation of STAT-3 in the human colorectal adenocarcinoma cell line DLD-1 and the gastric adenocarcinoma cell line MKN-28, and that antibodies of the invention can inhibit this IL-11-mediated phosphorylation. Panels a and B show the level of STAT-3 phosphorylation in DLD-1 and MKN-28 cells, respectively, after 15 minutes of stimulation with increasing concentrations of hIL-11. Panels C and D show the inhibition of IL-11 mediated STAT-3 phosphorylation in DLD-1 and MKN-28 cells by increasing concentrations of 8E2 anti-IL-11R antibody (O), respectively. In contrast, the BM4 isotype control antibody (. DELTA.) had no effect on IL-11 mediated STAT-3 phosphorylation. ( Antibody was added to the cells followed by stimulation with hIL-11 (50 ng/ml). Mean and standard deviation are shown. )
Detailed Description
SUMMARY
Throughout, unless otherwise indicated or otherwise required by context, a single step, composition of matter, group of steps, or group of compositions of matter shall be taken to include one or more (i.e., one or more) of these steps, composition of matter, group of steps, or group of compositions of matter.
It will be appreciated by those skilled in the art that the invention is susceptible to variations and modifications other than those described in the specification. It is to be understood that the present invention encompasses all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any two or more or any and all combinations of said steps or features.
The scope of the invention is not limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention.
Any embodiment of the invention will be mutatis mutandis to any other embodiment of the invention unless explicitly stated otherwise.
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry and biochemistry).
Unless otherwise indicated, recombinant proteins, cell culture and immunological techniques used in the present invention are standard methods, which are well known to those skilled in the art. Such techniques are described and explained in the literature, for example J.Perbal, A Practical Guide to Molecular Cloning (molecular cloning practice guide), john Welison Press (John Wiley and Sons) (1984), J.Sambrook et al, molecular Cloning: a Laboratory Manual (molecular cloning: laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbour Laboratory Press) (1989), T.A. Brown (ed.), essential Molecular Biology: a Practical Approach (Proprietary molecular biology: practice guidelines, volumes 1 and 2, IRL Press (1991), D.M.Glover and B.D.Hames (eds.), DNA Cloning: a PracticalApproach (DNA cloning: practice guidelines), volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al (eds.), current Protocols in Molecular Biology (New England Biolabs. Experimental guidelines), greene publishing Association and West science (1988, including all updates up to date), ed Harlow and David Lane (eds.), antibodies: a Laboratory Manual (antibodies: laboratory Manual), cold spring harbor laboratory Press (1988), and J.E. Coligan et al (eds.), current Protocols in Immunology (New Endochium of immunology), john Welisen Press (including all updates to today).
The descriptions and definitions of the variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein are further clarified by the discussion in the following documents: kabat Sequences of Proteins of Immunological Interest (immune protein sequences of interest), besseda, md., 1987 and 1991, bark et al, J mol. Biol.242, 309-320, 1994, chothia and Lesk J.mol biol.196:901-917, 1987, chothia et Al, nature342, 877-883, 1989 and/or Al-Lazikani et Al, J Mol Biol 273, 927-948, 1997.
The term "and/or" (e.g., "X and/or Y") is understood to mean "X and Y" or "X or Y" and should provide explicit support for both meanings or for each meaning.
Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The term "derived from" as used herein means that a particular integer may be obtained from a particular source, but need not be directly from that source.
The ranges mentioned herein (e.g., residues) are to be understood as inclusive. For example, a "region comprising amino acids 56-65" is to be understood in an inclusive manner, i.e., the region comprises sequences encoding amino acids 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in a particular sequence.
Definition of selection
For purposes of nomenclature only and not limitation, exemplary sequences of the human precursor IL-11Rα (IL-11 Rα precursor) are listed in NCBI reference sequence NP-001136256.1 (set forth in SEQ ID NO: 1). The sequence of mature human IL-11Rα is set forth in SEQ ID NO:100. in the case of the sequence listed in NP 001136256.1, the mature protein lacks amino acids 1-22. Amino acid positions are generally defined herein in terms of IL-11Rα precursors. The position in mature IL-11Rα can be readily determined by counting the signal sequence (amino acids 1-22 in the case of SEQ ID NO: 1). Exemplary sequences of cynomolgus monkey IL-11Rα precursors are set forth in SEQ ID NO:2 and mature IL-11 ra is set forth in SEQ ID NO:101. exemplary sequences of mouse IL-11Rα precursors are set forth in SEQ ID NO:82 and mature IL-11 ra is set forth in SEQ ID NO:102. the sequences of IL-11Rα from other species may be determined using the sequences provided by the present invention and/or publicly available databases and/or using standard techniques (see, e.g., ausubel et al (eds.), current Protocols in Molecular Biology (New Programming guidelines for molecular biology experiments), green publishing Association and Welch sciences or J.Sambrook et al, molecular Cloning: A Laboratory Manual (molecular cloning: laboratory guidelines), cold spring harbor laboratory Press (1989)). Human IL-11Rα is abbreviated hIL-11Rα, cynomolgus IL-11Rα is abbreviated cynomolgus IL-11Rα, and mouse IL-11Rα is abbreviated mIL-11Rα. Soluble IL-11Rα refers to a polypeptide comprising the extracellular region of IL-11Rα (e.g., amino acids 23-363 or 23-318 of SEQ ID NO: 1). In this study, a polypeptide comprising SEQ ID NO:1 (e.g., SEQ ID NO:3 or 85) and at a position corresponding to amino acids 23-363 or 23-318 of SEQ ID NO:82, the corresponding segment (e.g., SEQ ID NO 90) having a serine substituted mll-11 ra at position 206 was used for the study of mouse receptors. These serine mutations are introduced into soluble polypeptides to improve polypeptide expression and prevent polypeptide aggregation. Also used is SEQ ID NO:3 and SEQ ID NO:85 (see, e.g., SEQ ID NOS: 95-99). These soluble polypeptides are representative of hIL-11Rα or mIL-11Rα, as shown by the ability of the IL-11Rα binding proteins of the invention to bind to a related receptor when expressed on the cell surface. Thus, studies using mutant polypeptides are models of studies using hIL-11Rα and/or mIL-11Rα.
IL-11 herein includes both the native form of IL-11 and its mutated forms which retain the binding of IL-11R α (e.g. hIL-11R α ) And induces the ability to signal transduction.
hIL-11R herein α The specific domain of (a) is understood to mean the following:
immunoglobulin-like (IG-like) domain: SEQ ID NO:1 from amino acids 23 to 110;
a first fibronectin type III domain: SEQ ID NO: amino acids 111-215 of 1;
second fibronectin type III domain: SEQ ID NO: amino acids 216-370 of 1;
transmembrane domain: SEQ ID NO: amino acids 371-391 of 1; and
cytoplasmic domain: SEQ ID NO:1, amino acids 392-422.
The term "hIL-11 mutein" as used herein includes mutant forms of hIL-11 in which the wild-type residue at positions 58-62 (AMSAG) is replaced by PAIDY and tryptophan at residue 147 is replaced by alanine (W147A). For example, the mutein comprises the amino acid sequence of SEQ ID NO:110 or a combination thereof. Other examples of hIL-11 muteins can be found in WO2009/052588. Optionally, the mutein comprises an additional sequence, such as a six HIS tag.
The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide having the following characteristics: it is not related to the naturally associated components that accompany it in its natural state in its sense or derivative sense; substantially free of other proteins from the same source. Proteins can also be made substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. By "substantially purified" is meant that the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.
The term "recombination" is understood to mean the product of artificial genetic recombination. Thus, for recombinant proteins comprising an antibody antigen binding domain, the term excludes naturally occurring antibodies in a subject as a product of natural recombination occurring during B cell maturation. However, if such antibodies are isolated, they should be considered as isolated proteins comprising an antibody antigen binding domain. Similarly, if the nucleic acid encoding the protein is isolated and expressed using recombinant methods, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. Recombinant proteins also include proteins that are expressed by artificial recombinant means when they are within a cell, tissue or subject (e.g., a cell, tissue or subject in which the protein is expressed).
The term "protein" shall include a single polypeptide chain (i.e., a series of adjacent amino acids linked by peptide bonds) or a series of polypeptide chains (i.e., a polypeptide complex) that are covalently or non-covalently linked to each other. For example, the series of polypeptide chains may be covalently linked using a suitable chemical bond or disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.
The term "polypeptide" or "polypeptide chain" shall be understood from the preceding paragraphs to mean a series of adjacent amino acids linked by peptide bonds.
The term "antigen binding domain" as used herein is understood to mean an antibody region capable of specifically binding an antigen, i.e.V H Or V L Or comprises V H And V L Fv of (a) and (b). The antigen binding domain need not be in the whole antibody, e.g., it may be in an isolated form (e.g., a domain antibody) or in another form, e.g., as described above, such as an scFv.
For the purposes of the present invention, the term "antibody" includes proteins capable of specifically binding one or more closely related antigens (e.g., IL-11Rα) via an antigen binding domain contained within an Fv. The term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric, humanized, human, CDR-grafted, primate, deimmunized, humanized (synhumanized antibody), half-antibodies, bispecific antibodies). Antibodies typically comprise a constant region, which may be arranged as a constant region or constant fragment or crystallizable fragment (Fc). An exemplary form of an antibody comprises a four-chain structure as its basic unit. Full length antibodies comprise two heavy chains (about 50-70 kD) covalently linked to two light chains (about 23kDa each). The light chain typically comprises a variable region (if present) and a constant region and is either a kappa light chain or a lambda light chain in a mammal. Heavy chains typically comprise a variable region and one or two constant domains, which are linked to other constant domains by a hinge region. The mammalian heavy chain is one of the following types: alpha, 6, epsilon, gamma or mu. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains, as well as a heavy chain and a light chain, are immobilized together by interchain disulfide bonds and by non-covalent interactions. The number of interchain disulfide bonds may vary between different antibody types. Each chain has an N-terminal variable region (V H Or V L Each of about 110 amino acids in length) and one or more constant domains at the C-terminus. Constant domain of light chain (C L Of length of about 110 amino acids) and the first constant domain of the heavy chain (C H 1, which is 330-440 amino acids in length) are aligned and linked by disulfide bonds. Light chain variable region and heavy chain variable regionAlignment. The antibody heavy chain may comprise 2 or more other C' s H Domain (e.g. C H 2、C H 3, etc.) and may contain C H 1 and C H 2 constant domains. Antibodies can be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG) 1 、IgG 2 、IgG 3 、IgG 4 、IgA 1 And IgA 2 ) Or subclasses. In one embodiment, the antibody is a murine (mouse or rat) antibody or a primate (e.g., human) antibody. In one embodiment, the antibody heavy chain lacks a C-terminal lysine residue. In one embodiment, the antibody is humanized, chimeric, CDR-grafted or deimmunized.
The terms "full length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. In particular, whole antibodies include those having a heavy chain and a light chain (comprising an Fc region). The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.
As used herein, a "variable region" refers to a portion of the light and/or heavy chain of an antibody as defined herein that is capable of specifically binding an antigen and comprises the amino acid sequences of Complementarity Determining Regions (CDRs) (i.e., CDR1, CDR2, and CDR 3) and Framework Regions (FR). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR 4) and three CDRs. V (V) H Refers to the variable region of the heavy chain. V (V) L Refers to the variable region of the light chain.
The term "complementarity determining region" (synonymous CDRs; i.e., CDR1, CDR2 and CDR 3) as used herein refers to the amino acid residues of the variable region of an antibody, the presence of which is the primary cause of specific antigen binding. Each variable region domain (V H Or V L ) Typically there are three CDRs identified as CDR1, CDR2 and CDR3. In one embodiment, the amino acid positions assigned to the CDRs and FRs are according to Kabat et al, sequences of Proteins of Immunological Interest (protein sequences of immunological interest), national institutes of health (National Institutes of Health), besselda, malyland, 1987 and 1991(also referred to herein as "Kabat coding system"). In another embodiment, the amino acid positions assigned to the CDRs and FRs are defined according to the enhanced Chothia coding scheme (http:// www.bioinfo.org.uk/mdex. Html). According to Kabat coding system, V H The positions of FR and CDR are as follows: residues 1 to 30 (FR 1), 31 to 35 (CDR 1), 36 to 49 (FR 2), 50 to 65 (CDR 2), 66 to 94 (FR 3), 95 to 102 (CDR 3) and 103 to 113 (FR 4). According to Kabat coding system, V L The positions of FR and CDR are as follows: residues 1 to 23 (FR 1), 24 to 34 (CDR 1), 35 to 49 (FR 2), 50 to 56 (CDR 2), 57 to 88 (FR 3), 89 to 97 (CDR 3), and 98 to 107 (FR 4). The invention is not limited to the FR and CDR defined by the Kabat coding system, but includes all coding systems including canonical coding systems or Chothia and Lesk j.mol.biol.196:901-917, 1987; chothia et al, nature 342:877-883, 1989; and/or Al-Lazikani et Al, J.mol. Biol.273:927-948, 1997; honnegher and plurkthun j.mol.biol.309:657-670, 2001; or Giudielli et al, nucleic Acids Res.25: 206-2111997. In one embodiment, the CDRs are defined according to the Kabat coding system. Optionally, the heavy chain CDR2 according to the Kabat coding system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids is substituted with another naturally occurring amino acid. In this regard, padlan et al, FASEB j.,9:133-139, 1995 determined that the five C-terminal amino acids of heavy chain CDR2 are not normally involved in antigen binding.
"framework region" (FR) is the variable region residues other than the CDR residues.
The term "Fv" as used herein is understood to mean any protein, whether it consists of multiple polypeptides or a single polypeptide, wherein V L And V H Ligating and forming a complex having an antigen binding domain (i.e., capable of specifically binding an antigen). V forming an antigen binding domain H And V L May be co-located in a single polypeptide chain or in separate distinct polypeptide chains. In addition, fv's of the invention (as well as any protein of the invention) may have multiple antigen binding domains, which may or may not bind to the same antigen. The term should be understood to include directFragments derived from antibodies and proteins corresponding to such fragments produced using recombinant methods. In some embodiments, V H Is not associated with the heavy chain constant domain (C H ) 1 and/or V L Is not associated with the constant domain of the light chain (C L ) Are connected. Exemplary Fv-containing polypeptides or proteins include Fab fragments, fab 'fragments, F (ab') fragments, scFv, diabodies, triabodies, tetrabodies, or higher complexes, or with a constant region or domain thereof (e.g., C H 2 or C H 3 domain) linked to any of the foregoing, such as a miniantibody. "Fab fragments" consist of monovalent antigen binding fragments of immunoglobulins and can be produced by digestion of whole antibodies with papain to generate fragments consisting of intact light chains and a portion of heavy chains or can be produced using recombinant methods. Treatment of whole antibodies by pepsin followed by reduction to generate antibodies consisting of whole light chain and a portion of heavy chain (comprising V H And a single constant domain), a "Fab' fragment" of the antibody can be obtained. By this way, two Fab' fragments can be obtained per antibody. Fab' fragments can also be produced by recombinant methods. The "F (ab ') 2 fragment" of an antibody consists of a dimer of two Fab' fragments which are immobilized together by two disulfide bonds, and is obtained by treating the whole antibody molecule with pepsin without subsequent reduction. "Fab 2 A "fragment" is a recombinant fragment comprising two Fab fragments which are fused together using, for example, a leucine zipper or C H 3 domain linkage. A "single chain Fv" or "scFv" is a recombinant molecule comprising a fragment of a variable region (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
For interaction of an IL-11Rα binding protein or antigen binding domain thereof with an antigen, the term "bind" as used herein refers to the interaction being dependent on the presence or absence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures rather than to common proteins. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unbound "A") in a reaction containing labeled "A" and protein reduces the amount of labeled "A" bound to the antibody.
The term "specific binding" or "specifically binding" as used herein is understood to mean: the IL-11Rα binding proteins of the invention react or link more frequently, more rapidly, longer, and/or with higher affinity to a particular antigen or cell expressing IL-11Rα than to an alternative antigen or cell. For example, IL-11Rα binding proteins bind IL-11Rα (e.g., hIL-11Rα or a polypeptide comprising a region thereof, e.g., SEQ ID NO:3 or 85 polypeptide) with significantly higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than binding to other interleukin receptors or antigens that are commonly recognized by multiple reactive natural antibodies (i.e., naturally occurring antibodies known to bind multiple naturally occurring antigens in humans). In one embodiment of the invention, the polypeptide comprising SEQ ID NO:95, SEQ ID NO:3, or a polypeptide comprising a region thereof (e.g., the extracellular domain of hIL-11 ra) or SEQ ID NO:3 or 85. Generally, but not necessarily, references to binding refer to specific binding, and each term should be understood to provide explicit support for the other term.
The term "non-detectably bind" as used herein is understood to mean that the IL-11Rα binding protein (e.g., antibody) binds to the candidate antigen at a level of less than 10% or 8% or 6% or 5% above background. The background may be the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control antigen. Binding levels are detected using a biosensor assay (e.g., biacore) in which the IL-11 ra binding protein is immobilized and contacted with an antigen.
The term "non-significant binding" as used herein is understood to mean that the level of binding of the IL-11 ra binding protein of the invention to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., like a isotype control antibody) and/or the level of binding detected in the presence of a negative control polypeptide. Binding levels are detected using a biosensor assay (e.g., biacore) in which the IL-11 ra binding protein is immobilized and contacted with an antigen.
The phrase "reduced binding" or "binding at a lower level" as used herein in relation to an antigen is understood to mean that the IL-11Rα binding protein (e.g., antibody) binds to the antigen (e.g., a mutant of SEQ ID NO:3 as described herein, such as a mutant comprising the sequence set forth in SEQ ID NO: 95) with at least about 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold lower affinity as compared to the control epitope or antigen (e.g., SEQ ID NO: 3).
If the dissociation constant (K) for binding of IL-11Rα binding protein or antibody to polypeptide D ) Less than K binding of the protein or antibody to another polypeptide D The IL-11Rα binding protein or antibody is said to "preferentially bind" to the polypeptide. In one embodiment, if IL-11Rα binding protein or antibody and polypeptide binding affinity (K D ) The IL-11Rα binding protein or antibody is said to preferentially bind to another polypeptide at least 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold greater than the affinity of the protein or antibody for binding to the polypeptide.
The term "capable of binding hIL-11Rα and cynoIL-11Rα" as used herein is understood to mean that the IL-11Rα binding protein cross-reacts with hIL-11Rα and cynoIL-11Rα, i.e., binds to either protein.
For purposes of explanation and as will be understood by those of skill in the art based on the exemplary subject matter herein, the "affinity" in this specification refers to K of a protein or antibody D 。
For purposes of explanation and as will be understood by those of skill in the art based on the exemplary subject matter herein, "affinity" is at least about "should be understood to mean an affinity (or K D ) Equal to the listed values or higher (i.e., listed as lower for affinity), i.e., 2nM has an affinity higher than 3 nM. In other words, the term may be "X or less affinity", where X is a number listed herein.
“IC 50 Is to be understood as meaning IC 50 Equal to or less than the recited values (i.e., recited as an IC 50 Lower value of (2) in terms of IC, i.e., 2. Mu.g/ml 50 IC of more than 1. Mu.g/ml 50 . In other words, the term may be "X or lower IC 50 ", wherein X is a number as set forth herein.
The term "epitope" (synonymous with "antigenic determinant") as used herein is understood to refer to a region of IL-11Rα that is bound by an IL-11Rα binding protein comprising the antigen binding domain of an antibody. The term is not necessarily limited to the particular residues or structure to which the IL-11 ra binding protein contacts. For example, the term includes the region of amino acids that are contacted across the IL-11Rα binding protein and 5-10 (or more) or 2-5 or 1-3 amino acids outside of that region. In some embodiments, when the IL-11Rα binding protein is folded, the epitope comprises a series of discrete amino acids that are positioned in close proximity to each other, i.e., a "conformational epitope". It will be appreciated by those skilled in the art that the term "epitope" is not limited to a peptide or polypeptide. For example, the term "epitope" includes a chemically active set of surface molecules (e.g., sugar side chains, phosphoryl side chains, or sulfonyl side chains), and in certain embodiments, epitope determinants may have specific three-dimensional structural features and/or specific charge characteristics.
The term "competitive inhibition" is understood to mean that the IL-11Rα binding proteins (or antigen binding domains thereof) of the invention reduce or prevent binding of the listed antibodies or IL-11Rα binding proteins to IL-11Rα (e.g., to hIL-11Rα). This may be due to the fact that the IL-11Rα binding protein (or antigen binding domain) and the antibody bind to the same or overlapping epitopes. From the foregoing, it is apparent that IL-11 ra binding proteins need not completely inhibit antibody binding, but rather, they need only reduce binding by a statistically significant amount, e.g., by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the IL-11 ra binding protein reduces binding of the antibody by at least about 30%, more preferably by at least about 50%, more preferably by at least about 70%, more preferably by at least about 75%, more preferably by at least about 80% or 85%, and more preferably by at least about 90%. Methods of determining competitive inhibition of binding are known in the art and/or described herein. For example, the antibody is exposed to IL-11Rα in the presence or absence of an IL-11Rα binding protein. An IL-11Rα binding protein is considered to competitively inhibit antibody binding if it binds less in the presence of the protein than in the absence of the protein. In one embodiment, the competitive inhibition is not due to steric hindrance.
For two epitopes, "overlapping" is understood to mean that the two epitopes share a sufficient number of amino acid residues to allow the binding of an IL-11 ra binding protein (or antigen binding domain thereof) that binds one epitope to competitively inhibit the binding of an IL-11 ra binding protein (or antigen binding domain) that binds the other epitope. For example, an epitope that "overlaps" shares at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 amino acids.
The term "neutralisation" as used herein is understood to mean that the protein is able to block, reduce or prevent IL-11 mediated signal transduction in a cell by IL-11Rα. Methods of determining neutralization are known in the art and/or described herein.
The term "disorder" as used herein refers to disruption or interference of normal function, is not limited to any particular disorder, and includes diseases or disorders.
As used herein, an "IL-11-related disorder" refers to any disorder caused by or associated with an excess of IL-11 or cells expressing IL-11 or administration of IL-11. Such conditions can be readily determined by those skilled in the art. Exemplary disorders are described herein.
The term "preventing" as used herein includes administration of an IL-11Rα binding protein of the invention to stop or arrest the progression of at least one symptom of a disorder. The term also includes treating a patient in remission to prevent or arrest a relapse.
The term "treatment" as used herein includes administration of an IL-11Rα binding protein of the invention to reduce or eliminate at least one symptom of a particular disease or disorder.
The term "subject" as used herein is understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the object is a person.
Antibodies to
In one embodiment, the IL-11Rα binding protein described in any of the embodiments herein is an antibody.
Methods for generating Antibodies are known in the art and/or described in Harlow and Lane Antibodies: a Laboratory Manual (antibodies: laboratory Manual, cold spring harbor laboratory (1988)). Typically, in such methods, an IL-11 ra (e.g., hIL-11 ra) or a region thereof (e.g., extracellular region) or an immunogenic fragment or epitope thereof or a cell expressing or displaying the substance (i.e., immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal (e.g., mouse, chicken, rat, rabbit, guinea pig, canine, equine, dairy cow, goat, or pig). The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.
Polyclonal antibody production was monitored by sampling the blood of immunized animals at various points after immunization. If desired, one or more further immunizations may be administered to achieve the desired antibody titer. The procedure of boosting and potency determination is repeated until the appropriate potency is reached. Upon achieving the desired level of immunogenicity, the immunized animal is bled and serum isolated and stored, and/or the animal is used to produce monoclonal antibodies (mabs).
Monoclonal antibodies are one exemplary form of the antibodies of the invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous population of antibodies capable of binding to the same antigen (e.g., to the same epitope within an antigen). The term is not intended to limit the source of the antibody or the manner in which the antibody is made.
For mAb production, any of a variety of known techniques may be used, such as the methods exemplified in US4196265 or Harlow and Lane (1988) supra.
For example, an immunogen is used to immunize a suitable animal under conditions sufficient to stimulate antibody-producing cells. Rodents (e.g., rabbits, mice, and rats) are exemplary animals. Mice genetically engineered to express human antibodies, and for example, not to express murine antibodies, may also be used to generate antibodies of the invention (see, e.g., WO 2002/066630).
After immunization, somatic cells with antibody-producing potential, particularly B lymphocytes (B cells), are selected for the mAb generation protocol. These cells may be obtained from biopsies of spleen, tonsils or lymph nodes, or from peripheral blood samples. The B cells from the immunized animal are then fused with cells of immortal myeloma cells, which are typically derived from the same species as the animal immunized with the immunogen.
The hybrids are amplified by culturing in a selective medium containing reagents that prevent de novo synthesis of nucleotides in the tissue culture medium. Exemplary agents are aminopterin, methotrexate, and azaserine.
The amplified hybridomas are functionally selected for antibody specificity and/or titer, e.g., by flow cytometry and/or immunohistochemistry and/or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, spot immunoassays, etc.).
Alternatively, ABL-MYC technology (New cloning, madison 53713, wis.) was used to produce MAb secreting cell lines (see, e.g., largaaespada et al, J.Immunol. Methods.197:85-95, 1996).
Antibodies can also be produced or isolated by screening a display library (e.g., a phage display library), e.g., as described in US6300064 and/or US 5885793. For example, the inventors isolated fully human antibodies from phage display libraries.
As described herein, some IL-11Rα binding proteins of the invention that bind hIL-11Rα cross-react with cynoiIL-11 Rα and/or bind polypeptides comprising regions of hIL-11Rα that have been mutated and/or are absent or some mutated forms of hIL-11Rα. These properties can be used to generate antibodies or IL-11Rα binding proteins.
For example, using a polypeptide comprising SEQ ID NO:3 screening of phage display library to identify proteins binding to the polypeptide. The mutant form of the polypeptide to which the IL-11Rα binding protein binds is then not detectably used (e.g., wherein valine at position 117 corresponding to SEQ ID NO:1 is replaced with glutamic acid (e.g., comprising the sequence of SEQ ID NO: 95)) to remove the cross-reactive protein and/or the mutant form of the polypeptide to which the IL-11Rα binding protein binds is used (e.g., comprising the sequence of SEQ ID NO:97, 98 or 99) to isolate the protein with the correct cross-reactivity. Screening procedures for immunization of non-human mammals may also be referred to based on the foregoing.
In another embodiment, a polypeptide comprising the extracellular domain of cynoiil-11 ra (or the region corresponding to amino acids 23-215 or 110-215 of SEQ ID NO: 1) is used to immunize an animal or screen a phage display library and screen for identified IL-11 ra binding proteins and/or antibodies to identify a polypeptide that hybridizes to SEQ ID NO:3 and/or 85 polypeptide and/or hIL-11Rα have cross-reactivity.
In other embodiments, IL-11Rα or an extracellular region thereof (optionally, a mutated form of 8E2 or 8D10 or 8E4 binding) is contacted with one of the foregoing antibodies. The phage display library is then contacted with IL-11 ra or a region and phage expressing a protein that competes with the antibody for binding for selection.
In other embodiments, the chimeric protein comprises, for example, mouse IL-11 ra, wherein an epitope of interest from hll-11 ra replaces the corresponding mouse sequence. The chimeric protein is then used to immunize mice (which are less likely to induce an immune response against the mouse protein) and/or screen phage display libraries. The resulting antibodies/proteins are then screened to identify those that bind to hIL-11Rα (particularly at the site of interest) but not to mouse IL-11Rα.
The antibodies of the invention may be synthetic antibodies. For example, the antibody is a chimeric, humanized, human, humanized, primate, or deimmunized antibody.
Deimmunized, chimeric, CDR grafted, humanized, homohumanized, primate, human and composite IL-11Rα junctions Heteroprotein
The IL-11 ra binding protein of the invention may be a CDR-grafted protein comprising CDRs from an antibody of a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FR from a human antibody, or comprising CDRs from an antibody of another antibody type (e.g., another human antibody type) grafted onto or inserted into FR from one antibody type (e.g., one human antibody type). The term also includes complex IL-11 ra binding proteins comprising, for example, one or more CDR-grafted variable regions and one or more, for example, human variable regions, chimeric variable regions, humanized variable regions, or primate variable regions.
The IL-11Rα binding proteins of the invention may be humanized proteins.
The term "humanized protein" is understood to mean a protein comprising human-like variable regions (human-like variable region) comprising CDRs of an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into an FR from a human antibody (this antibody type falls within the class of "CDR-grafted antibodies"). The humanized IL-11Rα binding protein further comprises the following proteins: wherein one or more residues of the human protein are modified by one or more amino acid substitutions and/or one or more FR residues of the human protein are replaced by corresponding non-human residues. Humanized proteins may also comprise residues not found in human antibodies nor in non-human antibodies. Any other region of the protein (e.g., the Fc region) is typically human. Humanization may be performed using methods known in the art, such as US5225539, US6054297, US7566771 or US5585089. The term "humanized protein" also includes super-humanized proteins, such as described in US 7732578. The term also includes complex proteins comprising, for example, one or more humanized variable regions and one or more, for example, human, chimeric, homologous or primate variable regions.
The IL-11Rα binding protein of the invention may be a human IL-11Rα binding protein. The term "human protein" as used herein refers to a protein having variable and optionally constant antibody regions found in humans (e.g., in human germline or somatic cells or from libraries produced using such regions). The "human" protein may comprise amino acid residues that are not encoded by a human sequence, such as mutations introduced by random or site-directed mutagenesis in vitro (particularly mutations involving mutations or conservative substitutions in a small number of protein residues (e.g., 1, 2, 3, 4, or 5 of the residues of the protein). These "human proteins" need not be generated as a result of a human immune response, but may be generated using recombinant methods (e.g., screening phage display libraries) and/or by transgenic animals (e.g., mice) comprising constant and/or variable regions encoding human antibodies and/or using guided selection (e.g., as described in US 5565332). The term also includes affinity maturation forms of such antibodies. For the purposes of the present invention, a human antibody may be considered to comprise a protein comprising FR from the human antibody or FR containing sequences from the consensus sequence of human FR and wherein one or more CDRs are random or semi-random, as for example described in US6300064 and/or US 6248516.
An exemplary human IL-11Rα binding protein is an antibody comprising the following variable region pairs:
(i) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ii) Comprising SEQ ID NO: 38V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(iii) Comprising SEQ ID NO: 39V of the sequence set forth in SEQ ID NO. 39 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(iv) Comprising SEQ ID NO:40, V of the sequence set forth in SEQ ID NO. 40 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(v) Comprising SEQ ID NO: 41V of the sequence set forth in SEQ ID NO. 41 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vi) Comprising SEQ ID NO: 42V of the sequence set forth in SEQ ID NO. 42 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(vii) Comprising SEQ ID NO: 43V of the sequence set forth in SEQ ID NO. 43 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(viii) Comprising SEQ ID NO: 44V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(ix) Comprising SEQ ID NO: 45V of the sequence set forth in SEQ ID NO. 45 H And comprisesSEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(x) Comprising SEQ ID NO: 46V of the sequence set forth in SEQ ID NO. 46 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xi) Comprising SEQ ID NO:47, V of the sequence set forth in SEQ ID NO. 47 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xii) Comprising SEQ ID NO: 48V of the sequence set forth in SEQ ID NO. 48 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xiii) Comprising SEQ ID NO: 49V of the sequence set forth in SEQ ID NO. 49 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xiv) Comprising SEQ ID NO: 50V of the sequence set forth in SEQ ID NO. 50 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xv) Comprising SEQ ID NO: 51V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xvi) Comprising SEQ ID NO: 52V of the sequence set forth in SEQ ID NO. 52 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xvii) Comprising SEQ ID NO: 53V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xviii) Comprising SEQ ID NO: 54V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xix) Comprising SEQ ID NO: 55V of the sequence set forth in SEQ ID NO. 55 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xx) Comprising SEQ ID NO: 56V of the sequence set forth in SEQ ID NO. 56 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxi) Comprising SEQ ID NO: 57V of the sequence set forth in SEQ ID NO. 57 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxii) Comprising SEQ ID NO: 58V of the sequence set forth in SEQ ID NO. 58 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxiii) Comprising SEQ ID NO: 59V of the sequence set forth in SEQ ID NO. 59 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxiv) Comprising SEQ ID NO: 60V of the sequence set forth in SEQ ID NO. 60 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxv) Comprising SEQ ID NO: 61V of the sequence set forth in SEQ ID NO. 61 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxvi) Comprising SEQ ID NO: 62V of the sequence set forth in SEQ ID NO. 62 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxvii) Comprising SEQ ID NO: 63V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxviii) Comprising SEQ ID NO: 64V of the sequence set forth in SEQ ID NO. 64 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxix) Comprising SEQ ID NO: 65V of the sequence set forth in SEQ ID NO. 65 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxx) Comprising SEQ ID NO: 66V of the sequence set forth in SEQ ID NO. 66 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxi) Comprising SEQ ID NO: 67V of the sequence set forth in SEQ ID NO. 67 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxii) Comprising SEQ ID NO: 68V of the sequence set forth in SEQ ID NO. 68 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxiii) Comprising SEQ ID NO: 69V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxiv) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxv) Comprising SEQ ID NO:70, V of the sequence set forth in SEQ ID NO. 70 H And a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L ；
(xxxvi) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 6V of the sequence set forth in SEQ ID NO. 6 L ；
(xxxvii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 7V of the sequence set forth in L ；
(xxxviii) Comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 8V of the sequence set forth in SEQ ID NO. 8 L ；
(xxxix) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 9V of the sequence set forth in SEQ ID NO. 9 L ；
(xl) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 10V of the sequence set forth in seq id no L ；
(xli) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 11V of the sequence set forth in seq id no L ；
(xlii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 12V of the sequence set forth in SEQ ID NO. 12 L ；
(xliii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 13V of the sequence set forth in SEQ ID NO. 13 L ；
(xliv) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 14V of the sequence set forth in seq id no L ；
(xlv) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 15V of the sequence set forth in seq id no L ；
(xlvi) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 16V of the sequence set forth in seq id no L ；
(xlvii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 17V of the sequence set forth in SEQ ID NO. 17 L ；
(xlviii) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 18V of the sequence set forth in SEQ ID NO. 18 L ；
(xlix) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 19V of the sequence set forth in SEQ ID NO. 19 L ；
(l) Comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 20V of the sequence set forth in seq id no L ；
(li) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:21, V of the sequence set forth in SEQ ID NO. 21 L ；
(lii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 22V of the sequence set forth in SEQ ID NO. 22 L ；
(liii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:23, V of the sequence set forth in SEQ ID NO. 23 L ；
(liv) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:24, V of the sequence set forth in SEQ ID NO. 24 L ；
(lv) comprises the amino acid sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:25, V of the sequence set forth in SEQ ID NO. 25 L ；
(lvi) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:26, V of the sequence set forth in SEQ ID NO. 26 L ；
(vii) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 27V of the sequence set forth in seq id no L ；
(lviii) a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 28V of the sequence set forth in seq id no L ；
(lix) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 29V of the sequence set forth in seq id no L ；
(lx) comprises SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 30V of the sequence set forth in SEQ ID NO. 30 L ；
(lxi) comprises the amino acid sequence of SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 31V of the sequence set forth in seq id no L ；
(lxii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 32V of the sequence set forth in seq id no L ；
(lxiii) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO: 33V of the sequence set forth in seq id no L The method comprises the steps of carrying out a first treatment on the surface of the Or (b)
(lxiv) comprising SEQ ID NO:37, V of the sequence set forth in seq id no H And a polypeptide comprising SEQ ID NO:34, V of the sequence set forth in seq id no L 。
Optionally V H Is linked to a heavy chain constant region (e.g., an IgG4 heavy chain constant region). In one embodiment, the heavy chain constant region lacks a C-terminal lysine residue.
Optionally V L In phase with the constant region of the light chainAnd (3) connecting.
The IL-11Rα binding proteins of the invention may be humanized proteins. The term "humanized protein" refers to a protein produced by the method described in WO 2007/019620. The humanized IL-11 ra binding protein comprises a variable region of an antibody, wherein the variable region comprises an FR from a New World (New World) primate antibody variable region and a CDR from a non-New World primate antibody variable region. For example, the humanized IL-11 ra binding protein comprises a variable region of an antibody, wherein the variable region comprises an FR from a new world primate antibody variable region and a CDR from a mouse or rat antibody. In one embodiment, the humanized IL-11Rα binding protein is an IL-11Rα binding antibody in which one or both of the variable regions are humanized. The term also includes complex proteins comprising, for example, one or more humanized variable regions and one or more, for example, human variable regions or humanized variable regions or chimeric variable regions.
The IL-11Rα binding proteins of the invention may be primatized proteins. "primatized proteins" comprise variable regions derived from antibodies which are generated upon immunization of a non-human primate (e.g., cynomolgus monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to generate a primate antibody. An exemplary method for producing a primatized antibody is described in US6113898. The term also includes complex proteins comprising, for example, one or more primate materialized variable regions and one or more, for example, human variable regions or humanized variable regions or chimeric variable regions.
In one embodiment, the IL-11Rα binding proteins of the invention are chimeric proteins. The term "chimeric protein" refers to a protein in which the antigen binding domain is from a particular species (e.g., mouse, such as mouse or rat) or belongs to a particular antibody class or subclass, while the remainder of the protein is from a protein derived from another species (e.g., human or non-human primate) or belongs to another antibody class or subclass. In one embodiment, the chimeric protein is a chimeric antibody comprising V from a non-human antibody (e.g., a murine antibody) H And/or V L And the remainder of the antibody is derived from a human antibody. Chimeric proteins of this type Production is known in the art and can be achieved by standard methods (described for example in US6331415; US5807715; US 4815567 and US 4816397). The term also includes complex proteins comprising, for example, one or more chimeric variable regions and one or more, for example, human variable regions or humanized variable regions or chimeric variable regions.
The invention also relates to deimmunized IL-11Rα binding proteins, e.g., as described in WO2000/34317 and WO 2004/108158. Deimmunized antibodies and proteins have one or more epitopes (e.g., B cell epitopes or T cell epitopes) removed (i.e., mutated) thereby reducing the likelihood that the subject will mount an immune response against the antibody or protein. For example, the IL-11Rα binding proteins of the invention are analyzed to identify one or more B or T cell epitopes and one or more amino acid residues within the epitope are mutated to reduce the immunogenicity of the IL-11Rα binding protein.
As will be appreciated by those skilled in the art from the foregoing, a "composite" protein comprises one form of V H (e.g. human) and another form of V L (e.g., humanization). The invention explicitly includes all V H And V L Combinations of forms.
Proteins containing antibody binding domains
Single domain antibodies
In some embodiments, the antibodies of the invention are or comprise single domain antibodies (which may be used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable region of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (domatis, inc.) (wottham, massachusetts; see, e.g., US 6248516).
Diabodies, triabodies, tetrabodies
In some embodiments, the protein of the invention is or comprises a diabody, triabody, tetrabody or higher order protein complex, e.g., as described in WO98/044001 and/or WO 94/007921.
For example, a diabody is a protein comprising two linked polypeptide chains, each polypeptide chain comprising structure V L -X-V H Or V H -X-V L Wherein V is L Is an antibody light chain variable region, V H Is an antibody heavy chain variable region, X is a variable region comprising insufficient residues to permit V in a single polypeptide chain H And V L The linker being linked (or forming Fv) or being deleted and V of one polypeptide chain H V bound to another polypeptide chain L To form an antigen binding domain, i.e., to form an Fv molecule that is capable of specifically binding to one or more antigens. V in each polypeptide chain L And V H V in each polypeptide chain, which may be identical L And V H May be different, thereby forming a bispecific diabody (i.e., comprising two Fv's having different specificities).
Single chain Fv (scFv)
Those skilled in the art will appreciate that scFv comprises V in a single polypeptide chain H And V L Zone and V H And V L Polypeptide linker between (which promotes scFv to form the desired structure for antigen binding, i.e.V for single polypeptide chain H And V L Are linked to each other to form Fv). For example, the linker comprises more than 12 amino acid residues with (Gly) 4 Ser) 3 Which is one of the more advantageous linkers for scFV.
The invention also relates to disulfide stabilized Fv (or diav or dsFv) wherein a single cysteine residue is introduced into V H FR and V of (F) L And links cysteine residues by disulfide bonds to produce stable Fv.
Alternatively or additionally, the invention includes dimeric scFv, i.e. comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g. by a leucine zipper domain (e.g. derived from Fos or Jun). Alternatively, the two scFv are linked by a peptide linker long enough to allow the two scFv to form and bind to an antigen, as described in US 20060263367.
Heavy chain antibodies
Heavy chain antibodies are structurally different from many other antibody formats, and while they contain heavy chains, they do not contain light chains. Thus, these antibodies are also referred to as "heavy chain only antibodies". Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also known as IgNAR).
The variable regions found in naturally occurring heavy chain antibodies are commonly referred to as "V" in camelid antibodies HH The V-NAR in the domains "and IgNAR, thereby associating it with the heavy chain variable region (which is referred to as" V "in conventional 4-chain antibodies H Domain ") and the light chain variable region (which is referred to as" V ") present in conventional 4-chain antibodies L Domain ") are distinguished.
Heavy chain antibodies from camelids and variable regions thereof and methods for their production and/or isolation and/or use are generally described in, but not limited to, the following references: WO94/04678, WO 97/49505 and WO 97/49505.
Heavy chain antibodies and variable regions thereof from cartilaginous fish and methods of producing and/or isolating and/or using the same are generally described in, but not limited to, WO 2005/118629.
Other antibodies or proteins comprising antigen binding domains thereof
The invention also relates to other antibodies and proteins comprising antigen binding domains thereof, such as:
(i) "Key and Orifice" bispecific antibodies as described in US 5731168;
(ii) A heterologous coupling protein as described in US 4676980;
(iii) Heterologous conjugate proteins produced using chemical cross-linking agents, as described in US 4676980; and
(iv)Fab 3 (as described in EP 19930302894).
Mutation of proteins
The invention also provides an IL-11Rα binding protein or nucleic acid encoding an IL-11Rα binding protein that is at least 80% identical to the sequences disclosed herein. In one embodiment, the IL-11Rα binding protein or nucleic acid of the invention comprises a sequence that is at least about 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein, which protein specifically binds IL-11Rα as described in any of the embodiments herein.
Alternatively or additionally, the IL-11Rα binding protein comprises a V as described in any of the embodiments herein H Or V L At least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical CDRs (e.g., trisubstantial)CDRs) that specifically bind to IL-11 ra as described in any of the examples herein. In this regard, the inventors produced several antibodies having a variety of different sequences within their CDRs. Described herein are methods for determining binding of a protein to IL-11Rα.
For example, the inventors identified a group of IL-11 ra binding proteins that were at least 40% identical in their HCDR1 (and optionally amino acids in the N-terminal direction of HCDR 1) and another subset of proteins that were at least 80% identical in their HCDR1 according to the Kabat coding system.
The inventors have also identified a class of IL-11Rα binding proteins that are 77% identical in their HCDR2 according to the Kabat coding system and a subclass of IL-11Rα binding proteins that are at least about 82% identical in their HCDR2 according to the Kabat coding system.
As described herein, it is also known in the art that five C-terminal residues of the heavy chain CDR2 can be mutated to conservative or non-conservative amino acid substitutions (31% of the residues) (Padlan et al, FASEB J.9:133-139, 1995). Thus, a protein may comprise CDR2 that is at least about 47% identical to the heavy chain CDR2 sequences disclosed herein.
For example, the inventors identified a group of IL-11Rα binding proteins that were at least about 44% identical in their HCDR3 according to the Kabat coding system.
For example, the inventors identified a polypeptide comprising SEQ ID NO:37, V of the sequence set forth in seq id no H Which may be substituted without losing function or resulting in improved function. In one embodiment, the sequence that hybridizes to SEQ ID NO:37, the IL-11Rα binding protein comprises 1-11 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:37, the IL-11 ra binding protein comprises 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:37, the IL-11Rα binding protein comprises 3 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:37, the IL-11Rα binding protein comprises 4 amino acid substitutions.
In one embodiment, the sequence that hybridizes to SEQ ID NO:37, the IL-11Rα binding protein comprises 1-4 amino acid substitutions in CDR 3. For example, a sequence corresponding to SEQ ID NO:37, the IL-11Rα binding protein comprises 1 or 2 or 3 or 4 amino acid substitutions in CDR 3.
In one embodiment, the sequence that hybridizes to SEQ ID NO:37, the IL-11Rα binding protein comprises 1-3 amino acid substitutions in CDR 2. For example, a sequence corresponding to SEQ ID NO:37, the IL-11Rα binding protein comprises 1 or 2 or 3 amino acid substitutions in CDR 2.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: tryptophan at position 54 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: threonine at position 56 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: aspartic acid at position 57 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: tryptophan at position 57 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO:37 leucine at position 57.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: proline at position 99 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: glutamic acid at position 100 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO:37 leucine at position 100.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: aspartic acid at 101 position 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO:37 leucine at position 104.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least the sequence set forth in SEQ ID NO: arginine at position 104 of 37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least tryptophan at position 54, aspartic acid at position 56 and leucine at position 57, each relative to the sequence set forth in SEQ ID NO:37.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least tryptophan at position 54, aspartic acid at position 56 and leucine at position 57, each relative to the sequence set forth in SEQ ID NO:37.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37 comprising at least tryptophan at position 54, threonine at position 56 and leucine at position 57, each relative to the sequence set forth in SEQ ID NO:37.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37, which comprises at least proline at position 99, glutamic acid at position 100, aspartic acid at position 101 and leucine at position 104, each relative to SEQ ID NO:37.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:37, which comprises at least proline at position 99, leucine at position 100, aspartic acid at position 101 and arginine at position 104, each relative to SEQ ID NO:37.
for example, the inventors identified a group of IL-11Rα binding proteins that were at least about 45% identical in their LCDR1 and another subset of proteins that were about 54% identical in their LCDR1 according to the Kabat coding system.
For example, the inventors have also identified a class of IL-11Rα binding proteins that are at least about 55% or 56% identical in their LCDR3 according to the Kabat coding system.
For example, the inventors identified a polypeptide comprising SEQ ID NO: 5V of the sequence set forth in SEQ ID NO. 5 L Which may be substituted without losing function or resulting in improved function. In one embodiment, the sequence that hybridizes to SEQ ID NO:5, the IL-11Rα binding protein comprises 1-11 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:5, the IL-11Rα binding protein comprises 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:5, the IL-11 ra binding protein comprises 3 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:5, the IL-11 ra binding protein comprises 4 amino acid substitutions. For example, a sequence corresponding to SEQ ID NO:5, the IL-11Rα binding protein comprises 5 amino acid substitutions.
In one embodiment, the sequence that hybridizes to SEQ ID NO:5, the IL-11Rα binding protein comprises 1-4 amino acid substitutions in CDR 3. For example, a sequence corresponding to SEQ ID NO:5, the IL-11 ra binding protein comprises 1 or 2 or 3 or 4 amino acid substitutions in CDR 3.
In one embodiment, the sequence that hybridizes to SEQ ID NO:5, the IL-11Rα binding protein comprises 1-5 amino acid substitutions in CDR 1. For example, a sequence corresponding to SEQ ID NO:5, the IL-11 ra binding protein comprises 1 or 2 or 3 or 4 or 5 amino acid substitutions in CDR 3.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: valine at position 29 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: aspartic acid at position 30 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO:5 lysine at position 31.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: valine at position 33 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: glutamic acid at position 34 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: alanine at position 91 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: histidine at position 91 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: glutamic acid at position 91 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: aspartic acid at position 93 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: phenylalanine at position 93 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: serine at position 93 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least the sequence set forth in SEQ ID NO: glutamine at position 94 of 5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5, which comprises at least valine at position 29, aspartic acid at position 30, lysine at position 31, valine at position 33 and glutamic acid at position 34, each relative to SEQ ID NO:5.
In one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5, which comprises at least alanine at position 91, glutamic acid at position 92, aspartic acid at position 93 and glutamine at position 94, each relative to the sequence set forth in SEQ ID NO:5.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5, which comprises at least histidine at position 91, glutamic acid at position 92, phenylalanine at position 93 and glutamine at position 94, each relative to SEQ ID NO:5.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least histidine at position 91, glutamic acid at position 92 and glutamine at position 94, each relative to SEQ ID NO:5.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least histidine at position 91, glutamic acid at position 92 and glutamine at position 94, each relative to SEQ ID NO:5.
in one embodiment, the IL-11Rα binding protein of the invention comprises the amino acid sequence of SEQ ID NO:5 comprising at least glutamic acid at position 92, serine at position 93 and glutamine at position 94, each relative to SEQ ID NO:5.
In one embodiment, the nucleic acid of the invention comprises a sequence that is at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence set forth herein and encodes an IL-11 ra binding protein having the functions described in any of the embodiments herein. The invention also includes nucleic acids encoding the IL-11Rα binding proteins of the invention, which differ from the sequences exemplified herein by the degeneracy of the genetic code.
The percent identity of a nucleic acid or polypeptide is determined by GAP (Needleman and wunsch. Mol. Biol.48, 443-453, 1970) analysis (GCG program), where GAP creation penalty = 5 and GAP extension penalty = 0.3. The query sequence is at least 50 residues in length and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, two sequences are aligned over the entire length.
The invention also relates to nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding the IL-11Rα binding proteins described herein. "moderately stringent" is defined herein as hybridization and/or washing in 2 XSSC buffer, 0.1% (w/v) SDS, or equivalent at a temperature in the range of 45-65 ℃. "high stringency" is defined herein as hybridization and/or washing at a temperature of at least 65℃in 0.1 XSSC buffer, 0.1% (w/v) SDS, or lower salt concentration or equivalent. Specific levels of stringency herein include equivalent conditions that use wash/hybridization solutions other than SSC, as known to those of skill in the art. For example, methods for calculating the temperature at which each strand of a double-stranded nucleic acid dissociates (also referred to as the melting temperature or Tm) are known in the art. Similar (e.g., within 5℃or within 10 ℃) temperatures equal to the Tm of the nucleic acid are considered high stringency. Moderately stringent is considered to be within 10℃to 20℃or 10℃to 15℃of the calculated Tm of the nucleic acid.
The invention also relates to mutant forms of the IL-11Rα binding proteins of the invention comprising one or more conservative amino acid substitutions compared to the sequences set forth herein. In some embodiments, the IL-11Rα binding protein comprises 10 or fewer (e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1) conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having similar side chains and/or hydrophilicity (hydrophilicity) and/or hydrophilicity.
Amino acid residue families having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The water affinity index (hydropathic indices) is described, for example, in Kyte and dolittle j.mol.biol.,157:105-132, 1982, and hydrophilicity index (hydrophylic indices) is described in, for example, US4554101.
The invention also relates to non-conservative amino acid changes. For example, it is of particular interest to use one charged amino acid and to replace another charged amino acid with a neutral or positively charged amino acid. In some embodiments, the IL-11Rα binding protein comprises 10 or fewer (e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1) non-conservative amino acid substitutions.
In one embodiment, the mutation occurs in the FR of the antigen binding domain of the IL-11Rα binding proteins of the invention. In another embodiment, the mutation occurs in the CDR of the IL-11Rα binding protein of the invention.
An exemplary method of producing a mutant form of an IL-11Rα binding protein includes:
DNA mutagenesis (Thie et al, methods mol. Biol.525:309-322, 2009) or RNA mutagenesis (Kopsidas et al, immunol. Lett.107:163-168, 2006; kopsidas et al, BMC Biotechnology,7:18, 2007; and WO 1999/058661);
introducing a nucleic acid encoding a polypeptide into a mutator cell (multiplexer cell), such as XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);
DNA shuffling (e.g., as described in Stemmer, nature 370:389-91, 1994); and
site-directed mutagenesis, see for example Dieffnbach (code) and Dveksler (code) (published in PCR Primer: A Laboratory Manual (PCR Primer: handbook of laboratory), cold spring harbor laboratory, new York, 1995).
Exemplary methods for determining the biological activity of the mutant IL-11 ra binding proteins of the invention are apparent to those skilled in the art and/or described herein, e.g., antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, binding, dissociation, and therapeutic efficacy are described herein.
Constant region
The invention includes antibodies and/or IL-11Rα binding proteins described herein comprising a constant region of an antibody. This includes antigen binding fragments of antibodies fused to Fc.
The sequences for producing the constant regions of the proteins of the invention can be obtained from several different sources. In some embodiments, the constant region of the protein, or a portion thereof, is derived from a human antibody. The constant regions or portions thereof may be derived from any antibody type including IgM, igG, igD, igA and IgE and any antibody isotype including IgG1, igG2, igG3 and IgG 4. In one embodiment, the constant region is a human isotype IgG4 or a stabilized IgG4 constant region.
In one embodiment, the Fc region of the constant region has a reduced ability to induce effector function, e.g., as compared to a native or wild-type human IgG1 or IgG3 Fc region. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC). Methods of assessing the effector function level of an Fc region containing protein are known in the art and/or described herein.
In one embodiment, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), such as a human IgG4 Fc region. Suitable IgG4 Fc region sequences will be apparent to those skilled in the art and/or are available from publicly available databases (e.g., from national center for biotechnology information (National Center for Biotechnology Information)).
In one embodiment, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" is understood to mean an IgG4 constant region modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or the propensity to form half antibodies or half antibodies. "Fab arm exchange" refers to a type of protein modification to human IgG4 in which the IgG4 weight and the attached light chain (half molecule) are exchanged for a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule may require two different Fab arms (resulting in a bispecific molecule) that recognize two different antigens. Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents (e.g., reduced glutathione). An "half antibody" is formed when an IgG4 antibody dissociates to form two molecules (each molecule contains a single heavy chain and a single light chain).
In one embodiment, the stabilized IgG4 constant region comprises proline at position 241 of the hinge region of the system according to Kabat (Kabat et al, sequences of Proteins of Immunological Interest (protein sequence of immunological interest), washington d.c., U.S., health and human service, 1987 and/or 1991). This position corresponds to position 228 of the hinge region according to the EU coding system (Kabat et al, sequences of Proteins of Immunological Interest (immunologically interesting protein sequences), washington, U.S.A., health and human services, 2001 and Edelman et al, proc. Natl. Acad. USA,63, 78-85, 1969). In human IgG4, this residue is typically serine. After proline was used instead of serine, the IgG4 hinge region contained the sequence CPPC. In this regard, it will be understood by those skilled in the art that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which imparts mobility to the two Fab arms of the antibody. The hinge region comprises cysteine residues that are involved in the inter-heavy chain disulfide bond. The coding system according to Kabat, which is generally defined as extending from Glu226 to Pro243 of human IgG 1. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming the inter-heavy chain disulfide (S-S) bond in the same position (see, e.g., WO 2010/080538).
Other examples of stabilized IgG4 antibodies are the following antibodies: wherein arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU coding system) is replaced with lysine, threonine, methionine or leucine (e.g., as described in WO 2006/033386). Additionally or alternatively, the Fc region of the constant region may comprise residues selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at a position corresponding to 405 (EU coding system). Optionally, the hinge region comprises a proline (i.e., CPPC sequence) at position 241 (as described above).
In another embodiment, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region is an IgG1 Fc region comprising substitutions at one or more positions (selected from 268, 309, 330, and 331). In another embodiment, the Fc region is an IgG1 Fc region comprising deletions of one or more of the following variants E233P, L234V, L A and G236 and/or one or more of the following variants A327G, A330S and P331S (Armour et al, eur J Immunol.29:2613-2624, 1999; shields et al, J Biol chem.276 (9): 6591-604, 2001). Other examples of non-immunostimulatory Fc regions are described, for example, in Dall' Acqua et al, J immunol.177:1129-11382006; and/or Hezareh J Virol;75:12161-12168, 2001.
In another embodiment, the Fc region is a chimeric Fc region, e.g., comprising at least one C from an IgG4 antibody H 2 domain and at least one C from an IgG1 antibody H 3, wherein the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of: 240. 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU coding) (e.g. as described in WO 2010/085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.
Enhancing effector function
In one embodiment, the IL-11Rα binding proteins of the invention can induce effector function or enhanced effector function.
In the context of the present invention, "effector function" refers to those biological activities mediated by cells or proteins that bind to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region), which results in killing of the cells. Examples of effector functions induced by antibodies include complement dependent cytotoxicity; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody Dependent Cellular Phagocytosis (ADCP); and B cell activation.
In one embodiment, the IL-11Rα binding proteins of the invention bind IL-11Rα on the surface of a cell in a manner that induces effector functions (e.g., ADCC or CDC).
For example, the IL-11Rα binding protein maintains binding to IL-11Rα on the cell surface for a sufficient time to induce effector functions (e.g., ADCC and/or CDC).
In one embodiment, the IL-11 ra binding proteins of the invention are capable of inducing enhanced effector functions, for example by virtue of a modified Fc region or by virtue of comprising a region capable of binding immune effector cells. For example, the level of effector function is increased compared to the level induced by a human IgG1 or IgG3 Fc region. The enhanced effector function induced by the IL-11 ra binding proteins of the invention may lead to enhanced therapeutic or prophylactic effects, e.g. not only by blocking the effect of IL-11 ra but also by killing or depleting the cells responsible for the disorder, e.g. by killing autoreactive T cells.
In one embodiment, the Fc region of the IL-11Rα binding proteins of the invention is modified to increase the level of effector function induced thereby compared to an unmodified Fc region. Such modifications may be directed to the glycosylation and/or tertiary structure level and/or secondary structure level and/or amino acid level of the Fc region.
Those skilled in the art will appreciate that enhanced effector function may be demonstrated in any of a variety of ways, such as higher effector levels, longer effector durations, or faster effector rates.
In one embodiment, the Fc region comprises one or more amino acid modifications that enhance its ability to induce enhanced effector function. In one embodiment, the Fc region binds one or more fcγrs (e.g., fcγriii) with higher affinity. In one embodiment, the Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230. 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, according to the EU index encoding of Kabat. In one embodiment, the Fc region comprises the amino acid substitution S239D/I332E, encoded according to the EU index of Kabat. The Fc region has an approximately 14-fold increase in affinity for fcγriiia as compared to the wild-type Fc region, and the Fc region has an approximately 3.3-fold increase in ability to induce ADCC as compared to the wild-type Fc region. In one embodiment, the Fc region comprises the amino acid substitutions S239D/A330L/I332E, encoded according to the EU index of Kabat. The Fc region has an approximately 138-fold increase in affinity for fcγriiia as compared to the wild-type Fc region, and the Fc region has an approximately 323-fold increase in ability to induce ADCC as compared to the wild-type Fc region.
Other amino acid substitutions that increase the ability of the Fc region to induce effector function are known in the art and/or are described, for example, in US6737056 or US7317091.
In one embodiment, glycosylation of the Fc region is altered to increase its ability to induce enhanced effector function. In this regard, natural antibodies produced by mammalian cells typically comprise branched, bi-branched (biantennary) oligosaccharides which are typically linked to the C of the Fc region by N-linkages H Asn297 of domain 2 is linked. The oligosaccharides may comprise a variety of carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the bi-branched oligosaccharide structure. In some embodiments, the Fc region of the invention comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region, i.e., the Fc region is "nonfucosylated". Such variants may have improved ability to induce ADCC. The method of producing a nonfucosylated antibody comprises: the antibody or antigen binding fragment thereof is expressed in a cell line that fails to express alpha-1, 6-fucosyltransferase (FUT 8) (see, e.g., yumanne-Ohnuki et al, biotechnol. Bioengineer.87:614-622, 2004), in cells that express small interfering RNAs for FUT8 (see, e.g., mori et al, biotechnol. Bioengineer.,88:901-908, 2004), and in cells that fail to express Guanosine Diphosphate (GDP) -mannose 4, 6-dehydratase (GMD) (see, e.g., kanda et al, j. Biotechnol, 130:300-310, 2007). The invention also relates to the use of proteins with reduced levels of fucosylation, e.g. proteins produced in cell lines modified to express beta- (1, 4) -N-acetylglucosaminyl transferase III (GnT-III) (see e.g.) Et al, nat.Biotechnol.17：176-180，1999)。
Other methods include the use of cell lines that inherently produce antibodies capable of inducing enhanced Fc-mediated effector function (e.g., duck embryo-derived stem cells for the production of viral vaccines, WO2008/129058; avianRecombinant protein production in cells, WO 2008/142124).
IL-11Rα binding proteins of the invention also include those having a truncated oligosaccharide, for example, wherein the double branched oligosaccharide attached to the Fc region is truncated by GlcNAc. Such proteins may have reduced fucosylation and/or improved ADCC function. Examples of such proteins are described, for example, in US6602684 and US20050123546.
IL-11Rα binding proteins having at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated. Such proteins may have improved CDC function. Such proteins are described, for example, in WO1997/30087 and WO1999/22764.
The IL-11Rα binding protein may further comprise an Fc region capable of inducing enhanced CDC levels. For example, hybrids of IgG1 and IgG3 produce antibodies with enhanced CDC activity (Natsume et al, cancer Res.68:3863-3872.2008).
Additionally or alternatively, the IL-11Rα binding protein may be fused or conjugated to a protein that binds immune effector cells (e.g., an antibody variable region), such as by binding to CD3 or CD16.
Methods for determining effector function are known in the art. In one embodiment, use is made of 51 Cr release test, europium release test or 35 S release assay to assess ADCC activity level. In each of these assays, cells expressing IL-11Rα are incubated with one or more of the listed compounds for a period of time and under conditions sufficient to allow the compounds to be taken up by the cells. At the position of 35 In the S release test, cells can be contacted with 35 S-labeled methionine and/or cysteine are incubated for a period of time sufficient to allow incorporation of the labeled amino acids into the newly synthesized protein. Followed by the presence or absence of an IL-11Rα junctionHeteroprotein and cells are cultured in the presence of immune effector cells (e.g., PBMC and/or NK cells). Subsequent detection in cell culture Medium 51 Cr, europium and/or 35 An increase in the amount of S in the presence of protein as compared to the absence of protein indicates that the binding molecule/agent has an effector function. Exemplary publications disclosing assays for assessing protein-induced ADCC levels include helstrom et al, proc.Natl Acad.Sci.USA 83:7059-7063, 1986 and Bruggemann et al, j.exp. Med.166:1351-1361, 1987.
Other assays for assessing protein-induced ADCC levels include ACTI for flow cytometry TM Non-radioactive cytotoxicity assays (cell technology, inc.) or CytoTox (california, usa)Nonradioactive cytotoxicity assay (Promega, wisconsin, U.S.A.).
Alternatively or in addition, the effector function of an IL-11 ra binding protein is assessed by determining its affinity for one or more fcγrs, e.g. as described in US 7317091.
A C1q binding assay may also be performed to confirm that IL-11Rα binding proteins are capable of binding to C1q and inducing CDC. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163, 1996).
Other modifications
The invention also relates to other modifications to IL-11Rα binding proteins or antibodies comprising an Fc region or constant region.
For example, the antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody comprises an Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region for a nascent Fc region (FcRn). For example, the Fc region has a higher affinity for FcRN at a lower pH (e.g., about pH 6.0) to promote Fc/FcRN binding in the endosome. In one embodiment, the Fc region has an increased affinity for FcRn at about pH 6.0 compared to an affinity at about pH 7.4, which promotes re-release of Fc into the blood after cell cycling. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.
Exemplary amino acid substitutions include T250Q and/or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F (according to the EU coding system). Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.
Exemplary IL-11Rα binding proteins
Exemplary variable region containing IL-11Rα binding proteins produced by the inventors are described in Table 1.
Table 1: exemplary IL-11Rα binding protein sequences
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Protein production
In one embodiment, any of the embodiments of the IL-11Rα binding proteins are produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and/or as known in the art.
Recombinant expression
In another embodiment, any of the embodiments described herein of IL-11Rα binding proteins are recombinant.
In the case of recombinant proteins, nucleic acids encoding IL-11Rα binding proteins can be cloned into expression constructs or vectors and subsequently transfected into host cells, such as E.coli cells, yeast cells, insect cells, or mammalian cells (e.g., simian COS cells, chinese Hamster Ovary (CHO) cells, human Embryonic Kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein). Exemplary cells for expressing the protein are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these objectives are known in the art and are described, for example, in Ausubel et al (eds.), current Protocols in Molecular Biology (New Programming molecular biology laboratory Manual), green publishing Association and Wedeli science (1988, including all updates to today) or Sambrook et al, molecular Cloning: a Laboratory Manual (molecular cloning: laboratory Manual, cold spring harbor laboratory Press (1989). A variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are known in the art, see for example US 481657 or US5530101.
After isolation, the nucleic acid is inserted so that it is operably linked to a promoter in the expression construct or expression vector for further cloning (amplification of DNA) or for expression in a cell-free system or cell.
The term "promoter" as used herein is intended to be in its broadest sense and includes transcriptional regulatory sequences of genomic genes, including the TATA box or initiation elements necessary for precise transcription initiation, with or without other regulatory elements (such as upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter nucleic acid expression, for example in response to developmental and/or external stimuli, or in a tissue-specific manner. The term "promoter" is also used herein to describe a recombinant, synthetic or fused nucleic acid or derivative that confers, activates or enhances the expression of an operably linked nucleic acid. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter spatial and/or temporal expression of the nucleic acid.
The term "operably linked" as used herein refers to positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is under the control of the promoter.
A number of vectors are available for expression in cells. The carrier component generally includes, but is not limited to, one or more of the following: signal sequences, sequences encoding proteins (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. Suitable sequences for protein expression will be understood by those skilled in the art. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, ipp, or thermostable enterotoxin II), yeast secretion signals (e.g., invertase precursors, alpha factor precursors, or acid phosphatase precursors), or mammalian secretion signals (e.g., herpes simplex gD signals).
Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF 1), the micronuclear RNA promoter (U1 a and U1 b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV 40), the rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter; hybrid regulatory elements comprising a CMV enhancer/β -actin promoter or an immunoglobulin promoter or active fragment thereof. An example of a useful mammalian host cell line is the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney (293 or subcloned 293 cells for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary Cells (CHO).
Typical promoters suitable for expression in yeast cells, such as those selected from the group consisting of Pichia pastoris (Pichia pastoris), saccharomyces cerevisiae (Saccharomyces cerevisiae) and schizosaccharomyces pombe (s.pombe), include, but are not limited to: ADH1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.
Methods for introducing an isolated nucleic acid or an expression construct comprising an isolated nucleic acid into a cell for expression are known to those of skill in the art. The technique used for a given cell depends on known successful techniques. Methods of introducing recombinant DNA into cells include microinjection, DEAE-dextran mediated transfection, liposome-mediated transfection (such as by use of lipofectamine reagents (Ji Buke company (Gibco) of maryland, usa)) and/or cell transfection reagents (Ji Buke company of maryland, usa)), PEG-mediated DNA uptake, electroporation, and microprojectile bombardment (such as by use of DNA-coated tungsten or gold particles (Agracetus inc. Lu Site, wisconsin, usa)), and the like.
Host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F (sigma), minimal Essential Media (MEM) (sigma), RPM1-1640 (sigma) and Darbert Modified Eagle Medium (DMEM) (sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.
Separation of proteins
Methods of isolating proteins are known in the art and/or described herein.
When IL-11Rα binding proteins are secreted into the culture medium, the supernatant of such expression systems is typically first concentrated using a commercially available protein concentration filter, such as an amikacin (Amicon) or MP (Millipore Pellicon) ultrafiltration device. Protease inhibitors (e.g., PMSF) may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants. Alternatively or in addition, the supernatant may be filtered and/or separated from the cells expressing the protein, for example using continuous centrifugation.
IL-11Rα binding proteins produced from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing methods. These methods are known in the art and/or are described in WO99/57134 or Ed Harlow and David Lane (Ed.) Antibodies: a Laboratory Manual (antibodies: laboratory Manual, cold spring harbor laboratory (1988)).
It will also be appreciated by those skilled in the art that the protein may be modified to include a tag to facilitate purification or detection, for example a polyhistidine tag (e.g. hexahistidine tag) or an influenza virus Hemagglutinin (HA) tag or a simian virus 5 (V5) tag or a FLAG tag or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein comprising a six his tag is purified by the following method: contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on a solid or semi-solid support that specifically binds to the six His tag, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively or in addition, antibodies or ligands that bind to the tag are used in the affinity purification method.
Conjugates
In one embodiment, the IL-11Rα binding proteins of the invention are conjugated to a compound. For example, the compound is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of an IL-11 ra binding protein in a subject, and mixtures thereof.
Other compounds may bind directly or indirectly to the IL-11 ra binding protein (e.g., may contain a linker in the case of indirect binding). Examples of compounds include radioisotopes (e.g., iodine-131, yttrium-90, or indium-111), detectable labels (e.g., fluorophores or fluorescent nanocrystals or quantum dots), therapeutic compounds (e.g., chemotherapeutic or anti-inflammatory agents), colloids (e.g., gold), toxins (e.g., ricin or tetanus toxoid), nucleic acids, peptides (e.g., peptides that bind serum albumin), proteins (e.g., proteins comprising the antigen binding domain of an antibody or serum albumin), compounds that increase the half-life of IL-11 ra binding proteins in a subject (e.g., polyethylene glycol or other water-soluble polymers having such activity), and mixtures thereof. Exemplary compounds that can be coupled to the IL-11 ra binding proteins of the invention and methods for such coupling are known in the art and are described, for example, in WO2010/059821.
The IL-11Rα binding protein may be coupled to nanoparticles (for example, reviewed in Kogan et al Nanomedicine (Lond).2:287-306, 2007). The nanoparticle may be a metal nanoparticle.
The IL-11Rα binding protein may be contained in an antibody-targeted bacterial minicell (e.g., as described in PCT/IB 2005/000204).
Some exemplary compounds that can be coupled to the IL-11Rα binding proteins of the invention are listed in Table 2.
Table 2. Compounds for coupling.
Assay Activity of IL-11Rα binding proteins
Binding to IL-11Rα and mutants thereof
From the present invention, the skilled artisan will appreciate that some IL-11Rα binding proteins of the invention bind to the extracellular region of hIL-11Rα (as described herein) and bind to specific mutated forms of the extracellular region of hIL-11Rα (e.g., SEQ ID NO:3 or SEQ ID NO:85 with or without certain point mutations) and/or bind to human and cynomolgus IL-11Rα. Methods for assessing binding to proteins are known in the art, for example as described in Scopes (published: protein purification: principles and practice (protein purification: principles and practices), third edition, springer Verlag, 1994). Such methods typically involve immobilizing the IL-11Rα binding protein and contacting it with a labeled antigen. After washing to remove non-specifically bound proteins, the amount of tag and the resultant bound antigen are detected. Of course, the IL-11Rα binding protein may be labeled and the antigen immobilized. A translation type test (Panning-type assay) may also be used. Alternatively or in addition, surface plasmon resonance testing may be used.
The assays described above can also be used to detect proteins that bind to hIL-11Rα or its extracellular region (e.g., contained within SEQ ID NO: 3) or to SEQ ID NO:3 or SEQ ID NO:85 polypeptide or a mutant form thereof.
In one embodiment, the IL-11Rα binding proteins of the invention bind to the amino acid sequence of SEQ ID NO:95 polypeptide to SEQ ID NO:85 polypeptide binding level is at least about 1.5 times or 2 times or 5 times or 10 times or 50 times or 100 times or 150 times or 160 times or 200 times lower.
In one embodiment, the protein of the invention hybridizes to SEQ ID NO:96 polypeptide to SEQ ID NO:85 polypeptide binding level is at least about 1.5 times or 2 times or 5 times or 10 times or 50 times or 100 times or 150 times or 160 times or 200 times lower.
In one embodiment, the protein of the invention hybridizes to SEQ ID NO:86 polypeptide to SEQ ID NO:85 polypeptide binding level is at least about 1.5 times or 2 times or 5 times or 10 times or 50 times or 100 times or 150 times or 160 times or 200 times lower.
In one embodiment, the protein of the invention hybridizes to SEQ ID NO:89 polypeptide and SEQ ID NO:85 polypeptide binding level is at least about 1.5 times or 2 times or 5 times or 10 times or 50 times or 100 times or 150 times or 160 times or 200 times lower.
The level of binding is conveniently determined using a biosensor.
The present invention relates to any combination of the foregoing features. In one embodiment, the proteins described herein have all of the binding properties listed in the five paragraphs above.
Epitope mapping
In another embodiment, the epitopes bound by the proteins described herein are mapped. Epitope mapping methods will be apparent to those skilled in the art. For example, a series of overlapping peptides are produced that span the IL-11Rα sequence or region thereof that comprises an epitope of interest, such as a peptide comprising 10-15 amino acids. The IL-11Rα binding protein is then contacted with each peptide and the peptide bound thereto is determined. This allows the determination of peptides comprising epitopes to which the protein binds. If a protein binds to multiple non-contiguous peptides, the protein may bind to conformational epitopes.
Alternatively or in addition, amino acid residues within IL-11Rα are mutated, e.g., by alanine scanning mutagenesis or substitution of evolutionarily conserved amino acids, and mutations that reduce or prevent binding of IL-11Rα binding protein are determined. Any mutation that reduces or prevents binding of the IL-11Rα binding protein may be in an epitope to which the IL-11Rα binding protein binds.
In this aspect, as described herein, relative to SEQ ID NO: mutation of valine at position 117 of IL-11R of 1 reduces or prevents binding of 8E2 and 8D 10. Other tests of affinity maturation variants of 8E2 confirm that the V117 residue of IL-11R is more important for binding than other residues analyzed by mutation.
Another method of determining the region comprising the epitope to which the IL-11Rα binding protein binds involves substituting the region of hIL-11Rα with a corresponding region of the form of IL-11Rα to which the IL-11Rα binding protein does not bind (e.g., mIL-11Rα). If the IL-11Rα binding protein does not bind to the mutant form of IL-11Rα, residues forming part of the epitope of the protein may be within the region to be substituted.
Other methods for determining the epitope-containing region involve binding IL-11Rα or a region thereof to an immobilized IL-11Rα binding protein of the invention and digesting the resulting complex with a protease. Peptides that remain bound to the immobilized IL-11 ra binding protein are then isolated and analyzed (e.g., using mass spectrometry) to determine their sequences.
Other methods involve converting hydrogen in IL-11 ra or a region thereof to deuterons and binding the resulting protein to an immobilized IL-11 ra binding protein of the invention. The deuterons are then converted back to hydrogen, the IL-11 ra or regions thereof are isolated, consumed using an enzyme, and analyzed (e.g., using mass spectrometry) to identify those regions that contain deuterons that are protected from conversion to hydrogen due to binding of the IL-11 ra binding proteins described herein.
Optionally, determining the dissociation constant (Kd), the binding constant (Ka) and/or the affinity constant (K) of the immobilized IL-11 ra binding protein to IL-11 ra or an epitope thereof D ). In one embodiment, the "Kd" or "Ka" or "K" of the IL-11Rα binding protein is measured by a radiolabeled or fluorescently labeled IL-11Rα binding assay D ". In the case of "Kd", the assay balances IL-11Rα binding proteins using the lowest concentration of labeled IL-11Rα in the presence of a titration series (titration series) of unlabeled IL-11Rα. After washing to remove unbound IL-11 ra, the amount of tag was determined, which represents the Kd of the protein.
According to another embodiment, the method is performed by using a surface plasmon resonance test (e.g., using BIAcore surface plasmon resonance (Piscataway, N.J.)BIAcore Co.), to determine Kd, ka or K D Wherein an immobilized IL-11Rα or region thereof or an immobilized IL-11Rα binding protein is used.
In some embodiments, the IL-11Rα binding protein has a similar K as compared to antibody 8E2 D Or an improved KD (i.e., a lower KD value) because it may compete for binding to IL-11 ra.
Determination of competitive binding
Assays to determine proteins that competitively inhibit the binding of antibodies 8E2 and/or 8D10 and/or 8E4 will be apparent to those skilled in the art. One such method is exemplified herein.
For example, the antibody is conjugated to a detectable label, such as a fluorescent label or a radioactive label. The labeled antibodies are then mixed with a test IL-11Rα binding protein and contacted with IL-11Rα or a region thereof (e.g., contained within a polypeptide comprising SEQ ID NO: 3) or a cell expressing IL-11Rα or a region thereof. The level of the labeled antibody is then determined and compared to the level determined when the labeled antibody contacts the IL-11Rα, region or cell in the absence of the IL-11Rα binding protein. If the level of the labeled antibody is reduced in the presence of the test IL-11Rα binding protein as compared to the absence of the IL-11Rα binding protein, the IL-11Rα binding protein is considered to competitively inhibit binding of the antibody to IL-11Rα.
Optionally, the test IL-11 ra binding protein is conjugated to a different tag of the antibody. The surrogate markers allow for detection of the binding level of the test IL-11Rα binding protein to IL-11Rα or a region or cell thereof.
In another embodiment, the IL-11Rα binding protein is allowed to bind to IL-11Rα or a region thereof (e.g., contained within a polypeptide comprising SEQ ID NO: 3) or a cell expressing IL-11Rα or a region thereof, and then the IL-11Rα, region or cell is contacted with an antibody. A decrease in the amount of antibody bound in the presence of the IL-11 ra binding protein as compared to the absence of the IL-11 ra binding protein indicates that the antibody competitively inhibits binding of the antibody to IL-11 ra. A complementation assay (recrocal assay) can also be performed using a labeled IL-11Rα binding protein and allowing the antibody to bind IL-11Rα first. In this case, a decrease in the amount of labeled IL-11Rα binding protein that binds to IL-11Rα in the presence of the antibody as compared to the absence of the antibody indicates that the IL-11Rα binding protein competitively inhibits the binding of the antibody to IL-11Rα.
SEQ ID NO:3 and/or SEQ ID NO: any of the above assays are performed, for example, as described herein, with mutant forms of 85 and/or IL-11 ra and/or 8E2 and/or 8D10 and/or 8E4 bound extracellular regions.
Assay neutralization
In some embodiments of the invention, the protein is capable of neutralizing IL-11 signaling.
For assessing the ability of proteins and ligands to signal transduction through receptors, a variety of assays are known in the art.
In one embodiment, the protein reduces or prevents IL-11 from binding IL-11Rα. These assays can be performed in the form of the competitive binding assays described herein, wherein a labeled IL-11 and/or a labeled IL-11Rα binding protein is used.
In other embodiments, the IL-11Rα binding protein reduces proliferation of cells expressing IL-11Rα and gp130 (e.g., baF3 cells) (e.g., cells modified to express both proteins) that are cultured in the presence of IL-11. Culturing the cells (e.g., about 1x 10) in the presence of IL-11 (e.g., about 0.3ng/mL to about 5ng/mL (e.g., 0.3ng/mL or 0.5ng/mL or 5ng/mL (for hIL-11)) or about 1ng/mL to about 5ng/mL (e.g., 1ng/mL or 3ng/mL or 5 ng/mL) for mIL-11) and the presence or absence of the tested IL-11Rα binding protein 4 Individual cells). Methods for assessing cell proliferation are known in the art and include, for example, MTT reduction and/or thymidine incorporation. IL-11Rα binding proteins that reduce proliferation levels compared to levels observed in the absence of IL-11Rα binding proteins are believed to neutralize IL-11R signaling. IC was determined by testing various concentrations of IL-11Rα binding protein 50 I.e. the concentration at which 50% of the maximum inhibition of cell proliferation occurs. IC (integrated circuit) 50 Is 10 μg/ml or less. In one embodiment, the IC 50 9 μg/ml or less. For example, the IC 50 8 μg/ml or less. For example, the IC 50 7 μg/ml or less. For example, the IC 50 Is 6. Mu.g/ml or less. For example, the IC 50 Is 5 μg/ml or less. For example, the IC 50 Is 4. Mu.g/ml or less. For example, the IC 50 Is 3 μg/ml or less. In one embodiment, in connection with each of the foregoing embodiments, the IC 50 May be 10pg/ml or more or 10ng/ml or more.
Similar assays as described in the preceding paragraphs can be performed using B9 cells or T10 cells (Dams-Kozlowska et al, BMC Biotechnol,12:8, 2012; and Yokote et al, J AOAC,83:1053-1057, 2000). Proliferation can be measured by colorimetrically detecting a decrease in the tetrazolium compound 4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazol-1, 3-benzenedisulfonate (WST-1) in an assay using T10 cells.
In other embodiments, the ability of IL-11Rα binding proteins to inhibit IL-11 mediated erythropoiesis is assessed. For example, lin is allowed to stand in the presence or absence of IL-11Rα binding protein - CD34 + Cells (e.g., from cord blood) are contacted with IL-11. The amount of erythropoiesis is determined by detecting the number of cells expressing CD235a (e.g., using FACS). An IL-11Rα binding protein that reduces the number of cells expressing CD235a compared to the number of cells expressing CD235a in the absence of the IL-11Rα binding protein is believed to neutralize IL-11R signaling.
In other embodiments, the ability of IL-11Rα binding proteins to inhibit IL-11 mediated phosphorylation of STAT3 is assessed. For example, cells expressing IL-11Rα and gp130 are cultured in the presence of IL-11 and in the presence or absence of IL-11Rα binding protein. The level of STAT3 phosphorylation was then assessed by Western blot or FASC using antibodies specific for phosphorylated STAT 3. Exemplary experiments using FACS are described in Dams-Kozlowska et al, BMC Biotechnol,12:8,2012.
In another embodiment, the ability of an IL-11Rα binding protein to inhibit IL-11 mediated proliferation of cancer cells, such as gastric cancer cells or Acute Myelogenous Leukemia (AML) cells, is assessed. In these assays, cancer cells (e.g., AGN or MKN45 gastric cancer cells) are cultured in the presence of IL-11 and in the presence or absence of IL-11Rα binding protein. In the case of AML cells, the cells are also cultured in the presence of G-CSF. Cell proliferation is then measured using standard techniques, for example as described above and/or by assessing the formation of L-CFU in AML cells. Exemplary assays suitable for use in the present invention include Zhang et al, int J Biol sci, 8:383-393, 2012 and Kimura et al, leukemia,13:1018-1027, 1999.
Other methods of assessing neutralization of IL-11 signaling are contemplated by the present invention.
Measuring effector function
As described herein, some IL-11 ra binding proteins of the invention have reduced effector function or have effector function (or enhanced effector function). Methods for assessing ADCC activity are known in the art.
In one embodiment, use is made of 51 Cr release test, europium release test or 35 S release assay to assess ADCC activity level. In each of these assays, cells expressing IL-11R are incubated with one or more of the listed compounds for a period of time and under conditions sufficient to allow the compounds to be taken up by the cells. At the position of 35 In the S release test, cells expressing IL-11Rα can be contacted with 35 S-labeled methionine and/or cysteine are incubated for a period of time sufficient to allow incorporation of the labeled amino acids into the newly synthesized protein. The cells are then cultured in the presence or absence of the IL-11Rα binding protein and in the presence of immune effector cells, such as Peripheral Blood Mononuclear Cells (PBMC) and/or NK cells. Subsequent detection in cell culture Medium 51 Cr, europium and/or 35 The amount of S, the presence of the IL-11 ra binding protein with little or no change compared to the absence of the IL-11 ra binding protein, indicates that the protein has reduced effector function and increased amount (or increased compared to the presence of the IL-11 ra binding protein comprising an IgG1 Fc region) compared to the absence of the IL-11 ra binding protein (shows effector function or enhanced effector function). Exemplary publications disclosing assays for assessing protein-induced ADCC levels include helstrom et al, proc.Natl Acad.Sci.USA 83:7059-7063, 1986 and Bruggemann et al, j.exp. Med.166:1351-1361, 1987.
Other assays for assessing protein-induced ADCC levels include ACTI for flow cytometry TM Non-radioactive cytotoxicity assays (cell technology, inc.) or CytoTox (california, usa)Nonradioactive cytotoxicity assay (Promega, wisconsin, U.S.A.).
A C1q binding assay may also be performed to confirm that IL-11Rα binding proteins are capable of binding to C1q and inducing CDC. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163, 1996).
Determination of half-life
Some IL-11Rα binding proteins included in the invention have improved half-life, e.g., are modified to extend their half-life as compared to unmodified IL-11Rα binding proteins. Methods for assaying IL-11Rα binding proteins with improved half-lives will be apparent to those skilled in the art. For example, IL-11Rα binding proteins were evaluated for their ability to bind to neonatal Fc receptor (FcRn). In this regard, an increase in FcRn binding affinity increases the serum half-life of the IL-11 ra binding protein (see, e.g., kim et al, eur J immunol.,24:2429, 1994).
The half-life of the IL-11 ra binding proteins of the invention can also be measured by pharmacokinetic studies, e.g. according to Kim et al, eur J immunol.,24:542 The method described in 1994. In this way, radiolabeled IL-11Rα binding protein is injected intravenously into mice and plasma concentration is measured periodically as a function of time, for example, 3 minutes to 72 hours after injection. The clearance curve thus obtained should be biphasic, i.e. have an alpha-phase and a beta-phase. To determine the in vivo half-life of the protein, the clearance rate in the β -phase was calculated and compared to the wild-type or unmodified protein.
Assessing treatment efficacy
Assays for assessing the efficacy of a treatment are described above with respect to determining neutralization by an IL-11 ra binding protein.
In another embodiment, the efficacy of the protein in treating a disorder is assessed using an in vivo assay.
For example, IL-11Rα binding proteins are tested in animal models of arthritis. Exemplary models include the SKG germ line of mice (Sakaguchi et al Nature, 426:454-460), the rat type II collagen arthritis model, the mouse type II collagen arthritis model, or antigen-induced arthritis models in several species (Bendele J Musculoskel Neuron Interact;1 (4): 377-385, 2001). In these assays, arthritis was induced and the ability of the IL-11Rα binding protein to reduce one or more symptoms of arthritis (e.g., markers of joint inflammation and/or inflammation in synovial fluid) was evaluated. IL-11Rα binding proteins that reduce the symptoms of arthritis are believed to be useful in the treatment of such disorders or IL-11 mediated disorders (e.g., IL-11 mediated inflammatory disorders).
Additionally or alternatively, the IL-11 ra binding protein may be tested in a model of COPD, for example a model in which a non-human mammal (e.g. a rodent, such as a mouse) is exposed to cigarette smoke. Following exposure, the mammalian IL-11 ra binding protein is administered and the number of neutrophils and/or the level of pneumonia in the lung is predicted or assessed using standard techniques. IL-11Rα binding proteins that reduce the number of pneumonia and/or neutrophils are believed to be useful in the treatment of pneumonia or COPD or IL-11 mediated disorders (e.g., IL-11 mediated inflammatory disorders such as IL-11 mediated inflammatory lung disease).
Additionally or alternatively, IL-11Rα binding proteins may be tested in Th 2-inflammatory conditions (e.g., asthma or airway hyperresponsiveness). An exemplary model of allergic asthma is the mouse OVA model, e.g. Wang et al, j.immunol.165:2222 Described in 2000. After induction of inflammation, IL-11Rα binding proteins are administered to mice and evaluated for symptoms of asthma, such as eosinophil count, mucus secretion, and/or goblet cell proliferation in bronchoalveolar lavage fluid (BAL). Other asthma models are known in the art and include sheep models of inflammatory asthma, mouse models of allergic asthma as described in WO2002/098216 (e.g.induced by host dust mite proteins (Fattouh et al Am J Respir Crit Care Med: 314-321, 2005)), mouse models of severe asthma in which IL-5 and eosinophil chemotactic protein are overexpressed, or mice receiving tracheal instillation of poly-1-lysine which is hypersensitive to methacholine when administered in aerosol form (Homma et al Am J Physiol Lung Cell Mol Physiol 289: L413-L418, 2005).
Alternatively or in addition, IL-11Rα binding proteins can be tested in a model of cancer (e.g., gastric cancer). For example, the mouse homozygote of the Y757F mutant of gp130 (gp 130 Y757F/Y757F ) Gastric tumors occur (Jenkins et al, blood 109:2380-2388, 2007). Mice (e.g., eight week old mice) are treated with IL-11Rα binding protein and the weight and/or number of gastric polyps is assessed. IL-11Rα binding proteins that reduce polyp size and/or weight are believed to be useful in the treatment of cancer. Similar assays can be used to test the effect on rectal cancer, wherein gp130 is treated with Azoxymethane (AOM) followed by sodium dextran sulfate (DSS) Y757F/Y757F Mice (essentially as described in Greten et al, cell,118:285-296, 2004) were treated with IL-11Rα binding protein to induce rectal cancer.
Additionally or alternatively, IL-11Rα binding proteins, such as Li et al, oncol. Lett.3, may be tested in a model of cancer metastasis or cancer-related bone disease: 802-806, 2012.
Disorders to be treated
The present invention relates to the treatment or prevention of any condition in a subject caused or exacerbated by IL-11.
In one embodiment, the disorder is an autoimmune or inflammatory disorder.
In one embodiment, the autoimmune disorder is an autoimmune joint disorder, such as inflammatory arthritis, rheumatoid arthritis, or idiopathic arthritis (e.g., juvenile idiopathic arthritis). In one embodiment, the disorder is rheumatoid arthritis.
In one embodiment, the autoimmune disorder is an autoimmune intestinal disorder, such as an inflammatory intestinal disease, such as ulcerative colitis or crohn's disease.
In one embodiment, the autoimmune disorder is an autoimmune skin disorder, such as psoriasis.
In one embodiment, the inflammatory disorder is an inflammatory lung disorder, such as neutrophil infiltration-related lung disease. For example, the condition is asthma, chronic Obstructive Pulmonary Disease (COPD), rhinitis or allergy. In one embodiment, the disorder is asthma.
Other exemplary inflammatory conditions include infection-induced inflammation (e.g., mycobacterium tuberculosis-induced inflammation), gastritis (e.g., associated with gastric cancer), or inflammatory dermatitis (e.g., atopic dermatitis).
In one embodiment, the condition is a wasting condition, such as cachexia or sarcopenia. In one embodiment, the wasting disorder is cachexia. For example, the cachexia is associated with or caused by a condition selected from the group consisting of: rheumatoid arthritis, diabetes, heart disease, chronic kidney disease, chronic pneumonia, enteritis, inflammatory bowel disease, aging, sepsis or AIDS. In one embodiment, the cachexia is associated with or caused by cancer.
In one embodiment, the condition is a bone disorder, e.g., caused by inadequate bone formation and/or excessive bone metabolism. Exemplary bone disorders include bone resorption/damage caused by osteoporosis (including postmenopausal osteoporosis), bone fractures, cancer (e.g., metastatic bone cancer, myeloma, or paget's disease), and bone resorption/damage caused by cancer therapy (e.g., chemotherapy, hormone removal, or hormone inhibition).
In one embodiment, the disorder is cancer. Exemplary cancers include hematologic cancers, cancers of epithelial origin, gastric cancer, pancreatic cancer, liver cancer, osteosarcoma, endometrial cancer, and ovarian cancer.
In one embodiment, the subject is resistant to, unable to respond appropriately to, or is not suitable for treatment with another compound for treating the disorder. For example, a subject with an autoimmune or inflammatory disorder is resistant to, unable to respond appropriately to, or unsuitable for treatment with: corticosteroids and/or immunosuppressants and/or cyclophosphamide and/or methotrexate and/or anti-TNF antibodies or soluble TNF receptors and/or anti-CD 20 antibodies and/or anti-IL 6 antibodies and/or anti-CD 22 antibodies.
Composition and method for producing the same
In some embodiments, the IL-11 ra binding proteins described herein can be administered orally, parenterally, by inhalation spray, absorbed, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implanted reservoir (in a formulation containing a conventional non-toxic pharmaceutically acceptable carrier), or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion techniques.
Methods for preparing IL-11 ra binding proteins in suitable forms (e.g., pharmaceutical compositions) for administration to a subject are known in the art and include, for example, remington's Pharmaceutical Sciences (Remington pharmaceutical science) (18 th edition, mark publishing company (Mack Publishing Company), oiston, 1990) and u.s.pharmacopeia: national Formulary (United states pharmacopoeia: national formulary) (Mark publishing company, iston, pa., 1984).
The pharmaceutical compositions of the invention are particularly suitable for parenteral administration, such as intravenous administration or administration into cavities or cavities of joints or organs. Compositions for administration typically comprise a solution of the IL-11 ra binding protein dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous vehicles may be used, such as buffered saline and the like. The composition may contain pharmaceutically acceptable auxiliary substances required to mimic physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the IL-11Rα binding protein of the present invention in these formulations can vary widely, and is selected primarily based on factors such as fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous carriers such as mixed oils and ethyl oleate may also be used. Liposomes can also be used as carriers. The carrier may contain small amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.
After formulation, the IL-11Rα binding proteins of the invention are administered in a manner compatible with the dosage formulation and in a therapeutically/prophylactically effective amount. Each formulation may be readily administered in a variety of dosage forms, as described above for the type of injectable solution, but also includes other pharmaceutically acceptable forms, such as tablets, pills, capsules or other solid, suppository, pessary, nasal solution or spray, aerosol, inhalant, liposomal forms, etc. for oral administration. Pharmaceutical "slow release" capsules or compositions may also be used. Sustained release formulations are typically designed to administer a constant drug level over an extended period of time and can be used to deliver the IL-11 ra binding proteins of the invention.
WO2002/080967 describes compositions and methods of administering aerosolized compositions comprising antibodies useful in the treatment of, for example, asthma, which are also suitable for administration of the IL-11 ra binding proteins of the invention.
Combination therapy
In one embodiment, the IL-11Rα binding proteins of the invention are administered in combination with another compound for the treatment of a disorder described herein in a manner that incorporates or additional therapeutic steps or additional components of a therapeutic formulation.
For example, the other compound is an anti-inflammatory compound. Alternatively or in addition, the other compound is an immunosuppressant. Alternatively or additionally, the other compound is a corticosteroid, such as prednisone and/or prednisolone. Alternatively or additionally, the other compound is methotrexate. Alternatively or additionally, the other compound is cyclophosphamide. Alternatively or additionally, the other compound is mycophenolate mofetil. Alternatively or additionally, the other compound is an anti-CD 20 antibody (e.g., rituximab or ofatuzumab). Alternatively or additionally, the other compound is an anti-CD 22 antibody (e.g., epalizumab). Alternatively or additionally, the other compound is an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab) or a soluble TNF receptor (e.g., etanercept). Alternatively or additionally, the other compound is a CTLA-4 antagonist (e.g., abamectin, CTLA 4-Ig). Alternatively or in addition, the other compound is an anti-IL-6 antibody. Alternatively or in addition, the other compound is a BLys antagonist, such as an anti-BLys antibody (e.g., belimumab).
In another embodiment, the other compound is a chemotherapeutic agent or other agent for treating cancer.
In another embodiment, the protein described herein is administered before or after radiation therapy for the treatment of cancer.
Timing and dosage of administration
The appropriate dosage of the IL-11Rα binding proteins of the invention will vary depending on the particular IL-11Rα binding protein, the condition to be treated, and/or the subject being treated. Those skilled in the art are able to determine the appropriate dosage, for example, by starting from a dose that has not reached an optimum and varying the dosage stepwise to determine the optimum or useful dosage. Alternatively, to determine the appropriate dose for treatment/prophylaxis, data from cell culture assays or animal studies are used, where the appropriate dose is within the circulating concentration range of the ED50 containing the less toxic or non-toxic active compound. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. A therapeutically/prophylactically effective dose can be predicted initially by cell culture assays. The dose in the animal model can be formulated to achieve a formulation including IC 50 (i.e., the concentration or amount of compound that achieves half-maximal symptom inhibition) in the circulating plasma concentration range (as determined by cell culture). This information can be used to more accurately determine the dosage for a person. For example, plasma levels may be determined by high performance liquid chromatography.
In some embodiments, the methods of the invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.
The term "therapeutically effective amount" is the following amount: which upon administration to a subject in need of treatment improves the status and/or prognosis of the subject and/or reduces or inhibits symptoms of one or more of the clinical conditions described herein to a level below that observed and accepted as a clinical diagnosis or clinical profile of the condition. The amount to be administered to a subject depends on the particular nature of the condition to be treated, the type and stage of the condition being treated, the mode of administration, and the characteristics of the subject (e.g., general health, other diseases, age, sex, genotype, and weight). One skilled in the art can determine the appropriate dosage based on these and other factors. Thus, the term should not be construed as limiting the invention to a particular amount, such as the amount or weight of protein, but rather, the invention includes any amount of IL-11Rα binding protein sufficient to achieve the indicated results in a subject.
The term "prophylactically effective amount" as used herein is understood to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. It will be appreciated by those of skill in the art that such amounts will vary depending, for example, on the particular IL-11 ra binding protein and/or the particular subject and/or the type or severity or level of the disorder being administered and/or the propensity (genetic or otherwise) for the disorder to occur. Thus, the term should not be construed as limiting the invention to a particular amount, such as the amount or weight of IL-11Rα binding protein, but rather the invention includes any amount of IL-11Rα binding protein that is sufficient to achieve the indicated results in a subject.
For in vivo administration of the IL-11Rα binding proteins described above, the normal dose may vary from about 10ng/kg of body weight of the subject per day to up to about 100mg/kg of body weight of the subject per day or higher. For repeated administrations over several days or longer, depending on the severity of the disease or condition to be treated, the treatment may be maintained until the desired suppression of symptoms is achieved.
In some embodiments, the IL-11Rα binding protein is administered at an initial (or loading) dose of about 1mg/kg to about 30mg/kg (e.g., about 1mg/kg to about 10mg/kg, or about 1mg/kg or about 2mg/kg or 5 mg/kg). IL-11Rα binding protein may then be administered at a lower maintenance dose of about 0.01mg/kg to about 2mg/kg (e.g., about 0.05mg/kg to about 1mg/kg, e.g., 0.1mg/kg to about 1mg/kg, e.g., about 0.1mg/kg or 0.5mg/kg or 1 mg/kg). The maintenance dose may be administered every 7-30 days (e.g., every 10-15 days, e.g., every 10 or 11 or 12 or 13 or 14 or 15 days).
In some embodiments, IL-11Rα binding protein is administered at a dose of about 0.01mg/kg to about 50mg/kg (e.g., about 0.05mg/kg to about 30mg/kg, e.g., about 0.1mg/kg to about 20mg/kg, e.g., about 0.1mg/kg to about 10mg/kg, e.g., about 0.1mg/kg to about 2 mg/kg). For example, IL-11Rα binding protein is administered at a dose of about 0.01mg/kg to about 5mg/kg (e.g., without a higher loading dose or a lower maintenance dose), such as about 0.1mg/kg to about 2mg/kg, such as about 0.2mg/kg or 0.3mg/kg or 0.5mg/kg or 1mg/kg or 1.5 mg/kg. In some embodiments, multiple doses are administered, e.g., once every 7-30 days, such as every 10-22 days, e.g., once every 10-15 days, such as every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, the IL-11Rα binding protein is administered once every 7 days or every 14 days or every 21 days.
In some embodiments, the IL-11 ra binding protein is administered to the mammal at the beginning of the treatment for no more than 7 consecutive days or 6 consecutive days or 5 consecutive days or 4 consecutive days.
Multiple doses may be administered within a week in the event that the mammal does not respond appropriately to the treatment. Alternatively or in addition, an ascending dose may be administered.
In another embodiment, for mammals experiencing adverse effects, the initial (or loading) dose may be divided into days of the week or consecutive days.
The administration of the IL-11Rα binding proteins according to the methods of the invention may be continuous or intermittent, depending, for example, on the physiological state of the recipient (whether the administration is for therapeutic or prophylactic purposes) and other factors known to those of skill in the art. The administration of the IL-11Rα binding protein may be substantially continuous over a predetermined period of time, or may be a series of spaced doses, for example during or after the development of the disorder.
IL-11Rα assay
The following assays can be performed using the IL-11Rα binding proteins of the invention (e.g., IL-11Rα binding proteins described herein coupled to a detectable label). Detection of IL-11Rα or cells expressing IL-11Rα using the assays described herein is useful for diagnosing or prognosticating a disorder.
Immunoassays are exemplary assay formats for diagnosing a disorder in a subject or detecting IL-11Rα or cells expressing IL-11Rα in a sample. The present invention relates to any immunoassay format, including Western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescent-linked immunosorbent assay (FLISA), competition assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescent assay, turbidimetric assay-based assay, nephelometric assay-based assay, and Fluorescence Activated Cell Sorting (FACS) based assay.
One form of suitable immunoassay is, for example, ELISA or FLISA.
In one form, such assays involve immobilizing the IL-11Rα binding proteins of the invention on a solid substrate, such as a polystyrene or polycarbonate microwell or impregnated strip, a membrane, or a glass support (e.g., a glass slide). The test sample is then contacted directly with the IL-11Rα binding protein and the IL-11Rα or IL-11Rα expressing cells in the sample are bound or captured. After washing to remove any unbound proteins in the sample, the IL-11Rα binding proteins that bind to IL-11Rα at different epitopes or to different antigens on the cells are brought into direct contact with the captured IL-11Rα or cells. The detection protein is typically labeled with a detectable reporter, which in ELISA is, for example, an enzyme such as horseradish peroxidase (HRP), alkaline Phosphatase (AP) or 3-galactosidase, and in FLISA is a fluorophore. Alternatively, a second labeled protein that binds to the detection protein may be used. After washing to remove any unbound protein in the sample, the detectable reporter is detected by adding a substrate, such as hydrogen peroxide, TMB or toluidine or 5-bromo-4-chloro-3-indole-beta-D-galactoside (x-gal) in ELISA. Of course, the immobilized (capture) protein and the detection protein may be used in the opposite manner.
The level of antigen in the sample is then determined using a standard curve, which has been generated using known amounts of the marker, or by comparison to a control sample.
The above assays can be readily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.
Those skilled in the art will appreciate that other detection methods based on immunoadsorption assays may be used to carry out the invention. For example, the immunoadsorption method or acridine immunoadsorption assay described above using a radioactive label for detection or a gold label for detection (e.g., colloidal gold) or a liposome (e.g., encapsulating nad+) for detection.
In some embodiments of the invention, the level of IL-11Rα or cells expressing IL-11Rα is determined using the following manner: surface plasmon resonance detector (e.g. BIAcore TM GE healthcare group (GE healthcare), piscataway, N.J.; flow-through devices (as described in US 7205159); micro-or nano-immunoassay devices (as described in US 20030124619); a flow measuring device (as described in US20040228761 or US 20040265926); fluorescence polarization immunoassay (FPIA, as described in US4593089 or US 4751190); or an immunonephelometry test (as described in US5571728 or US 6248597).
Sample and control sample
It will be appreciated by those of skill in the art that some of the embodiments described herein require a degree of quantification to determine the level of IL-11Rα or cells expressing IL-11Rα. Such quantification can be determined by inclusion of a suitable control sample in the assays of the invention.
In one embodiment, a suitable control sample is a sample derived from a healthy subject or a normal subject.
In this context, the term "healthy subject" is understood to mean an individual known not to suffer from an IL-11Rα related disorder (e.g. an inflammatory disorder).
The term "normal subject" is understood to mean an individual having normal IL-11Rα or IL-11Rα expressing cells levels in a sample as compared to a population of individuals.
The invention also includes control samples in the form of data sets obtained from normal and/or healthy subjects or populations of normal and/or healthy subjects.
In one embodiment, the methods of the invention further comprise determining IL-11Rα levels in a control sample, e.g., using the methods described above.
In one embodiment, the sample from the subject and the control sample are measured at about or substantially the same time.
In one embodiment, the same method described in any one or more embodiments of the invention is used to determine a sample from a subject and a control sample to allow comparison of the results.
Medicine box
The invention also includes a kit comprising one or more of the following:
(i) The IL-11Rα binding proteins of the invention or expression constructs encoding the proteins;
(ii) The cells of the invention; or (b)
(iii) The pharmaceutical composition of the invention.
In kits for detecting IL-11Rα, the kit may further comprise a detection method, e.g., in conjunction with an IL-11Rα binding protein of the invention.
In kits for therapeutic/prophylactic applications, the kit may further comprise a pharmaceutically acceptable carrier.
Optionally, the kit of the invention is provided with instructions for use in the method described in any of the examples.
The invention includes the following non-limiting examples.
Non-limiting examples
Materials and methods
Phage display
Human IL-11Rα -specific antibodies were isolated from a library of human Fab-phagemid antibodies. Phage that bound to immobilized hIL-11Rα -Fc (R & D Systems) catalog number 1977-MR) were eluted in the presence of wild-type hIL-11 or hIL-11 muteins. Multiple positive clones (confirmed by phagemid ELISA) were reconstituted to human IgG4 antibodies.
IL-11 reactive cell proliferation assay
hIL-11, cynoIL-11 and mIL-11 responsive BaF3 cell lines were used to test antibodies for their ability to block IL-11 bioactivity. The mIL-11 reactive BaF3 cell line is described in WO2009/052588.
hIL-11 and cynomoIL-11-reactive BaF3 cell lines were stably transfected with constructs encoding wild-type human IL-11Rα and cynomolgus 11Rα, respectively, and human gp130 and cynomolgus gp130, respectively. The cells were selected by growth in media containing hIL-11 or cynoiIL-11 and cloned by limiting dilution cloning to obtain a clonal cell line. Dose-responsive proliferation (using a thymidine incorporation assay) of multiple stably transfected clones was analyzed when cultured in the presence of either hIL-11 or cynoiL-11. One hIL-11 reactive clone and one cynoiIL-11 reactive clone were selected for analysis of antibodies.
IL-11-reactive BaF3 cells were treated at about 1X 10 in the presence of a sub-maximum concentration of IL-11 (hIL-11: 0.3ng/mL or 0.5ng/mL or 5ng/mL; mIL-11:1ng/mL, 3ng/mL or 5ng/mL; cyno IL-11:0.5ng/mL or 5 ng/mL) and increasing concentrations of purified monoclonal antibody or hIL-11 mutein (comprising SEQ ID NO:110 and having an N-terminal six HIS tag) in a total volume of 200. Mu.L/well 4 Individual cells/wells were seeded in 96-well plates containing RPMI/10% fcs/Glutamax/PenStrep. The cells were initially incubated for 30 minutes in the presence of antibodies or hIL-11 muteins, after which cytokines were added. The cells were incubated at 37℃for about 48-50 hours and used for the last 6 hours of incubation 3 H-thymidine was pulsed. Cells were harvested onto glass fiber filter plates and the level of radioactive thymidine incorporated into DNA was determined by liquid scintillation counting. The test was repeated twice and then plotted using the average of the individual test points.
Affinity maturation of antibody 8E2
Antibody 8E2 was affinity matured using the following method. V of 8E2 to be encoded H And V L Is inserted into the phagemid construct to encode the germline Fab and a germline termination template is created by replacing 18 codons (6 amino acid residues) in all CDRs (except CDR-L2) with TAA termination codons. Libraries were constructed using the method essentially as described by Sidhu et al (Methods in Enzymology:238:333-336, 2000). Each termination template was used as a template for the Kunkel mutagenesis method (Kunkel et al, methods in Enzymology:154:367-382, 1987), in which the mutagenized oligonucleotides were designed to repair the termination codon simultaneously and introduce mutations at the designed sites. The mutagenesis reaction was introduced into E.coli by electroporation and phage production was initiated by the addition of helper phage. After overnight growth at 30 ℃, phage were harvested using PEG/NaCl precipitation.
Using decreasing concentrations of the nucleic acid sequences comprising SEQ ID NO:3 by cycling the library through several rounds of selection. The target concentration was reduced by a factor of 10 in each round.
Binding of antibodies to domain-exchanged IL-11R mutant polypeptides
Various soluble forms of IL-11R comprising regions of hIL-11R and mIL-11R are produced and comprise the amino acid sequence of SEQ ID NO:3 and 86-90, and determining binding of antibodies 8E2, 8D10 and 8E4 to those polypeptides using Western blot or Biacore analysis.
For Biacore analysis, anti-human IgG was immobilized on the sensor surface at about 10,000-12,000RU and each antibody was captured at 0.2 μg/ml to 0.5 μg/ml per cycle for 120 seconds. IL-11 receptor was injected at various concentrations (from 1. Mu.M down to 2.5 nM) on the captured antibodies, including the injection of buffer blank. Binding was monitored for 3-5 min and dissociation was monitored for 3-10 min. By fitting the binding curve for each interaction to 1:1 kinetic model to determine the approximate affinity.
Binding of antibodies to Point mutants of soluble hIL-11R
Multiple point mutants of soluble forms of IL-11R were generated in which amino acids from mIL-11 were introduced into soluble forms of hIL-11 (SEQ ID NO: 85) (for mutation positions relative to SEQ ID NO:1 and SEQ ID NO: for each polypeptide, see Table 5) and antibodies were assayed against those polypeptides or SEQ ID NO:3 or SEQ ID NO:85 binding of the polypeptide.
Anti-human IgG Fc specific antibodies were chemically immobilized on CM5 chips to about 9000RU. Antibodies were captured at about 0.3-1 μg/ml for 120 seconds at 2 points in each flow cell. Adjacent spots consisting only of anti-human IgG were used as references. IL-11 receptor injections at 40, 10, 5 and 2.5nM were repeated twice on captured antibodies and reference points. Blank injections of buffer alone were also repeated twice. Injection was continued for 3 minutes and dissociation was monitored for an additional 5 minutes.
The binding curves were subtracted from the control and buffer was removed before fitting to the 1:1 kinetic model.
Antibody-mediated inhibition of IL-11 signaling in cancer cells
DLD-1 (colorectal cancer cell line) and MKN-28 (gastric cancer cell line) cells were stimulated with increasing concentrations of hIL-11 for 15 min or incubated in the presence of increasing concentrations of 8E2 anti-IL-11R or BM4 isotype control antibodies followed by stimulation with huIL-11 (50 ng/ml). Cells were fixed and permeabilized, followed by staining with PE-conjugated anti-phosphostat-3 antibody. Cells were analyzed by flow cytometry. The test was repeated in duplicate.
Example 1: antibody isolation and characterization
Three antibodies 8E2, 8D10 and 8E4 were isolated from the Fab-phagemid antibody library based on their ability to inhibit proliferation of hIL-11 dependent transfected BaF3 cells. 8E4 also inhibited mouse IL-11 dependent BaF3 cell proliferation.
8E2, 8D10 and 8E4 bind to cells transfected with hIL-11Rα or cynoiL-11Rα.8E4 (but not 8E2 and 8D 10) was bound to cells transfected with mIL-11Rα.
Of these three antibodies, 8E2 had the best thermal stability (Tm was 69.8-76.6 ℃ C., while 8D10 and 8E4 were 63.2-71.4 ℃ C.) as assessed by differential scanning calorimetry.
8E2 was selected and affinity maturation was performed. Heavy and light chain libraries were used to generate clones with mutated CDRs (fig. 1, 2 and 3). 62 of these affinity variants were assessed for binding to hIL-11R by Biacore and the relative ability to inhibit hIL-11-induced proliferation of BaF3 cells was determined. The results are shown in Table 3.
Table 3: characterization of affinity matured antibodies
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1 Antibody V H And V L The sequences of (2) are listed in Table 1
2 Efficacy (g/ml) by BaF humanIL-11R cell proliferation assay
3 Biacore data were generated at pH 8.5 and 37℃
NP indicates no efficacy; N/D indicates not measured
* Some of the heterogeneity observed makes it difficult to determine the exact K D
Ten affinity matured antibodies (TS-306, TS-2, TS-4, TS-7, TS-14, TS-51, TS-101, TS-108, TS-134, and TS-136) were selected based on improved ability to inhibit hIL-11-induced proliferation of BaF3 cells compared to 8E2, 8D10, and 8E4, and improved affinity to human IL-11R compared to 8E 2. CDR sequences are also considered in selecting these affinity matured antibodies. Several clones were selected that had CDR sequences close to or identical to the relevant consensus sequence.
These antibodies were tested against proliferation of human, cynomolgus and/or mouse IL-11 induced BaF3 cells transfected with human, cynomolgus or mouse IL-11R. Binding to human and cynomolgus IL-11R transfected cells was also determined and the average fluorescence intensity was measured by flow cytometry. Each selected clone inhibited hIL-11 and cyno-IL-11 induced proliferation more strongly and bound human and cynomolgus IL-11R with higher affinity than the parental 8E2 clone.
Example 2: comparison of antagonism of hIL-11 muteins with antibodies
Several antibodies were compared for antagonistic activity against hIL-11 muteins (comprising SEQ ID NO:110 and having an N-terminal six HIS tag) using the assay described above, employing the BaF3 cell line transfected with nucleic acid encoding human IL-11R and human gp130 and 0.3ng/mL human IL-11. The results are shown in Table 4.
Table 4: comparison of IL-11 Signal transduction antagonism between anti-IL 11R antibodies and hIL-11 muteins
The data shown in Table 4 demonstrate that all antibodies tested were more potent than hIL-11 muteinsInhibiting IL-11 signaling. IC due to the large difference in molecular weight between muteins and antibodies 50 Values are expressed in nM.
Example 3: epitope mapping
The competition ELISA and Biacore data showed that 8E2, 8E4 and 8D10 compete with each other for binding to hIL-11Rα and can recognize the same region of hIL-11Rα.
Domain exchange (mutein-human) Biacore data shows that at least amino acids 111-215 (Fn 3-type domain 1) of hIL-11 ra (SEQ ID NO: 1) are required for 8E2 and 8D10 binding, and that the use of a mouse sequence instead of a human sequence in this region reduces the affinity of 8E4 binding (table 5).
TABLE 5 binding of IL-11 receptor to antibodies
N/A: unevaluated
N/B: no binding or binding fall-off
4E5 is a mouse monoclonal antibody, which has strong binding affinity to mouse IL-11R.
In the results shown in Table 5, "strong binding" means affinity (K D ) About 10nM or less. "weak binding" refers to affinity (K D ) About 50nM or higher.
28 constructs with single amino acid substitutions in the region covering Ig-like domain of hIL-11Rα and fibronectin type 3 domain 1 were constructed by replacing the natural human amino acid with the corresponding mouse amino acid residue. Five mutants of truncated hIL-11R (SEQ ID NO: 85) (P65S, K R, L101S, V117E, A178S) (encoded relative to the amino acid position in SEQ ID NO: 1) were expressed and purified. The corresponding constructs were used to transfect cells, which were subsequently stained using 8E2, 8E4 and 8D 10. 8E2 and 8E10 binding was reduced in V117E transfected cells as demonstrated by flow cytometry.
The streaming data is confirmed by Biacore. All Biacore sensorgrams fit well to the 1:1 binding model and derived K D The values are shown in Table 6.
The V117E mutant did not bind to 8E2 and 8D10 at the concentrations used in this assay and the K of this interaction could not be determined D . This residue is involved in the binding interaction of the two antibodies.
The K66R mutation results in K for antibodies 8E2 and 8D10 D The decrease was doubled compared to WT D1/2 alone (SEQ ID NO: 85), indicating that this residue is involved in antibody binding to some extent.
Mutants V117E and K66R also bound antibody 8E4 with significantly lower affinity than WT D1/2 (SEQ ID NO: 85). These residues may be involved in the binding interaction of antibody 8E4 with hIL-11Rα.
ANOVA and Tukey multiple comparison tests showed that V95E and K44R affinities were significantly different from WT D1/2 (SEQ ID NO: 85) (p < 0.05) for all antibodies tested.
All of the mutant forms of IL-11R tested in this example bind IL-11, indicating that the mutation does not induce significant conformational changes in the receptor.
Table 6: affinity of antibodies to Point mutants of soluble human IL-11Rα (SEQ ID NO: 85)
Affinity of antibodies to various receptor mutants. For WT D1/2 (SEQ ID NO: 85) n=2, both values are shown. All other values are mean ± SE. N=3 for WT F/L (SEQ ID NO: 3) and n=4 for others. The bold displayed values have a significantly different K than WT D1/2 D Value (p < 0.05). The position of the mutation relative to SEQ ID NO:1.* Indicating undetectable binding.
Example 4: comparing data
Non-neutralizing anti-IL-11R antibody (4D 12, a commercially available antibody from St. Cruz Co.) was demonstrated to bind human IL-11Rα by ELISA. It was demonstrated that 4D12 was combined with BaF3 cells transfected with human IL-11R alpha and with 293 cells transfected with human or mouse IL-11R alpha.
4D12 did not neutralize human or mouse IL-11 induced cell proliferation in BaF3 cells incubated with 0.5ng/mL hIL-11 or 3ng/mL mIL-11R.
Competitive ELISA was used to demonstrate that 4D12 did not compete with 8D10 or 8E2 for binding to IL-11 ra.
Example 5: anti-IL-11R antibodies inhibit IL-11 signaling in cancer cells
As shown in FIG. 4, IL-11 induced STAT-3 phosphorylation in DLD-1 colon cancer cells and MKN-28 stomach cancer cells. Figure 4 also shows that antibody 8E2 inhibits STAT-3 phosphorylation in these cells, whereas isotype control antibody does not.

Claims (11)

1. An IL-11 ra binding protein comprising an antigen binding domain of an antibody that binds or specifically binds IL-11 ra and neutralizes IL-11 signaling and that binds an epitope comprising residues within a first fibronectin type III domain of IL-11 ra, wherein the first fibronectin type III domain is amino acids 111-215 of SEQ ID NO 1, and wherein the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
The VH comprises: (i) Complementarity Determining Region (CDR) 1 of SEQ ID NO. 79, CDR2 of SEQ ID NO. 80, and CDR3 of SEQ ID NO. 81; and is also provided with
The VL comprises: (i) CDR1 of SEQ ID NO. 77, CDR2 of amino acids 50-56 of SEQ ID NO. 5, and CDR3 of SEQ ID NO. 78.
2. The IL-11 ra binding protein of claim 1, wherein:
the VH is SEQ ID NO: 71, and the VL is SEQ ID NO: 35; or (b)
The VH is SEQ ID NO: 72 and the VL is SEQ ID NO: 36.
3. The IL-11 ra binding protein of claim 1 or 2, wherein, if the V H And V L Co-located in a single polypeptide chain, then the protein is:
(i) Single chain Fv fragments (scFv);
(ii) Dimeric scFv (di-scFv);
(iii) With the constant region Fc or heavy chain constant domain (C) H ) 2 and/or C H 3 to one of (i) or (ii); or (b)
(iv) One of (i) or (ii) linked to a protein that binds immune effector cells, or
If said V H And V L In separate polypeptide chains, the protein is then:
(i) A diabody;
(ii) A tri-antibody;
(iii) A four-antibody;
(iv)Fab；
(v)F(ab’) 2 ；
(vi)Fv；
(vii) With the constant region Fc or heavy chain constant domain (C) H ) 2 and/or C H 3 to one of (i) to (vi);
(viii) One of (i) to (vi) linked to a protein that binds immune effector cells; or (b)
(ix) An antibody.
4. The IL-11 ra binding protein of claim 3, which is an antibody.
5. A nucleic acid encoding the IL-11 ra binding protein of any one of claims 1-4.
6. An expression construct comprising the nucleic acid of claim 5.
7. An isolated or recombinant cell that expresses the IL-11 ra binding protein of any one of claims 1-4.
8. A composition comprising the IL-11 ra binding protein of any one of claims 1-4 and a pharmaceutically acceptable carrier.
9. Use of an IL-11 ra binding protein according to any one of claims 1-4 or a composition according to claim 8 in the manufacture of a medicament for the treatment of an IL-11 mediated disorder.
10. The use of claim 9, wherein the IL-11 mediated disorder is an autoimmune disorder, an inflammatory disorder, a wasting disorder, a bone disorder, or cancer.
11. An in vitro method of detecting IL-11 ra or cells expressing IL-11 ra in a sample, the method comprising contacting the sample with the IL-11 ra binding protein of any one of claims 1-4 to form a complex and detecting the complex, detection of the complex being indicative of the presence of IL-11 ra or cells expressing IL-11 ra in the sample.
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Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017049349A1 (en) * 2015-09-25 2017-03-30 Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research A method of treatment and prophylaxis
GB201522186D0 (en) 2015-12-16 2016-01-27 Singapore Health Services Pte Ltd And Nat University Of Singapore The Treatment of fibrosis
CN109071613A (en) * 2016-05-11 2018-12-21 通用电气医疗集团生物工艺研发股份公司 Isolation medium
GB201621431D0 (en) 2016-12-16 2017-02-01 Singapore Health Services Pte Ltd And Nat Univ Of Singapore The Decoy cytokine receptor
GB201621439D0 (en) * 2016-12-16 2017-02-01 Singapore Health Services Pte Ltd And Nat Univ Of Singapore IL-11Ra Antibodies
MX2019007020A (en) 2016-12-16 2019-10-21 Singapore Health Serv Pte Ltd Il-11 antibodies.
GB201716733D0 (en) 2017-10-12 2017-11-29 Nat Univ Singapore Treatment of SMC mediated disease
GB201809700D0 (en) 2018-06-13 2018-08-01 Singapore Health Serv Pte Ltd IL-11 antibodies
KR20210138579A (en) 2019-01-21 2021-11-19 싱가포르 헬스 서비시즈 피티이 엘티디 Treatment of hepatotoxicity
GB201902419D0 (en) 2019-02-22 2019-04-10 Singapore Health Serv Pte Ltd Treatment of kidney injury
GB201906291D0 (en) 2019-05-03 2019-06-19 Singapore Health Serv Pte Ltd Treatment and prevention of metabolic diseases
MX2021013356A (en) * 2019-05-03 2022-03-11 Singapore Health Serv Pte Ltd Treatment and prevention of metabolic diseases.
GB202009292D0 (en) 2020-06-18 2020-08-05 Singapore Health Serv Pte Ltd Treatment and prevention of disease caused by type IV collagen dysfunction
EP3939999A1 (en) * 2020-07-14 2022-01-19 Fundación del Sector Público Estatal Centro Nacional de Investigaciones Oncológicas Carlos III (F.S.P. CNIO) Interleukin 11 receptor alpha subunit (il11ra) neutralizing antibodies and uses thereof
GB202017244D0 (en) 2020-10-30 2020-12-16 Nat Univ Singapore Methods to extend health-span and treat age-related diseases
CN117157317A (en) 2021-02-26 2023-12-01 拜耳公司 Inhibitors of IL-11 or IL-11Ra for the treatment of abnormal uterine bleeding
WO2023006765A1 (en) 2021-07-26 2023-02-02 Boehringer Ingelheim International Gmbh Treatment and prevention of alcoholic liver disease
IL311043A (en) * 2021-08-30 2024-04-01 Lassen Therapeutics 1 Inc Anti-il-11rα antibodies
CN113735975B (en) * 2021-09-07 2022-08-12 广东东阳光药业有限公司 anti-IL-11R antibody and application thereof
WO2023111196A1 (en) 2021-12-16 2023-06-22 Singapore Health Services Pte. Ltd. Treatment and prevention of glomerular disease

Family Cites Families (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4196265A (en) 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
US4593089A (en) 1980-07-30 1986-06-03 Abbott Laboratories Fluorescent polarization immunoassay utilizing substituted triazinylaminofluorescein aminoglycosides
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US4751190A (en) 1985-07-22 1988-06-14 Abbott Laboratories Fluorescence polarization immunoassay and reagents for use therein
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
WO1990005144A1 (en) 1988-11-11 1990-05-17 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
DE4211351A1 (en) 1992-04-04 1993-10-07 Behringwerke Ag Method for analyzing particle-enhanced agglutination reactions on centrifugal analyzers by determining the turbidity brightening
DK1589107T3 (en) 1992-08-21 2010-04-26 Univ Bruxelles Immunuglobulins without light chains
WO1994007921A1 (en) 1992-09-25 1994-04-14 Commonwealth Scientific And Industrial Research Organisation Target binding polypeptide
US6274547B1 (en) * 1996-06-14 2001-08-14 Genetics Institute, Inc. Methods comprising the use of human interleukin-11 receptor proteins
EP1566440A3 (en) * 1994-12-22 2005-10-19 Genetics Institute, Inc. Human Interleukin-11 Receptor
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
AUPO591797A0 (en) 1997-03-27 1997-04-24 Commonwealth Scientific And Industrial Research Organisation High avidity polyvalent and polyspecific reagents
US6113898A (en) 1995-06-07 2000-09-05 Idec Pharmaceuticals Corporation Human B7.1-specific primatized antibodies and transfectomas expressing said antibodies
AU725609C (en) 1995-08-18 2002-01-03 Morphosys Ag Protein/(poly)peptide libraries
GB9603256D0 (en) 1996-02-16 1996-04-17 Wellcome Found Antibodies
US6541213B1 (en) 1996-03-29 2003-04-01 University Of Washington Microscale diffusion immunoassay
CA2258518C (en) 1996-06-27 2011-11-22 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recognition molecules interacting specifically with the active site or cleft of a target molecule
CA2244326C (en) 1997-08-11 2006-03-28 Shinichi Eda Microparticle enhanced light scattering agglutination assay and microparticle reagents therefor
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6489447B1 (en) 1998-05-06 2002-12-03 Genentech, Inc. Protein purification
US6562622B1 (en) 1998-05-08 2003-05-13 Diatech Pty, Ltd Continuous in vitro evolution
CA2237915A1 (en) * 1998-05-19 1999-11-19 Stephen Shaughnessy Osteoporosis treatment
ES2278463T3 (en) 1998-12-08 2007-08-01 Biovation Limited METHOD TO REDUCE THE IMMUNOGENICITY OF PROTEINS.
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US6566051B1 (en) 1999-01-15 2003-05-20 Medtox Scientific, Inc. Lateral flow test strip
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
EP1355919B1 (en) 2000-12-12 2010-11-24 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
US8178098B2 (en) 2001-04-03 2012-05-15 National Jewish Health Method to inhibit airway hyperresponsiveness using aerosolized T cell receptor antibodies
AUPR544401A0 (en) 2001-06-04 2001-06-28 Bischof, Robert Juergen Animal model for allergy
MXPA05000511A (en) 2001-07-12 2005-09-30 Jefferson Foote Super humanized antibodies.
JP4264348B2 (en) 2001-08-20 2009-05-13 プロテオム システムズ リミテッド Diagnostic inspection method and apparatus
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
AU2003302132B2 (en) 2002-11-21 2008-02-21 Inverness Medical Switzerland Gmbh Bodily fluid markers of tissue hypoxia
US20050014716A1 (en) 2003-05-21 2005-01-20 Wang Jin Wei Pancreatic cancer treatment
US20070292416A1 (en) 2003-06-02 2007-12-20 Alexion Pharmaceuticals, Inc. De-Immunized Anti-Cd3 Antibody
US20070190024A1 (en) * 2003-07-29 2007-08-16 Inserm Institut National De La Sante Et De La Rech Il-11 muteins
TR201809892T4 (en) 2003-11-05 2018-07-23 Roche Glycart Ag Antigen binding molecules with binding affinity to the Fc receptor and enhanced effector function.
DK2330121T3 (en) 2004-06-02 2014-12-15 Adalta Pty Ltd Binding moieties based on shark IgNar domains
ES2387312T3 (en) 2004-09-22 2012-09-20 Kyowa Hakko Kirin Co., Ltd. Stabilized human IgG4 antibodies
US20070135620A1 (en) 2004-11-12 2007-06-14 Xencor, Inc. Fc variants with altered binding to FcRn
US20060263367A1 (en) 2005-05-23 2006-11-23 Fey Georg H Bispecific antibody devoid of Fc region and method of treatment using same
CA2619244A1 (en) 2005-08-15 2007-02-22 Arana Therapeutics Limited Engineered antibodies with new world primate framework regions
CN101321538A (en) * 2005-12-06 2008-12-10 惠氏公司 Interleukin-11 compositions and methods of use
EP1985305A1 (en) 2007-04-24 2008-10-29 Vivalis Duck embryonic derived stem cell lines for the production of viral vaccines
EP1995309A1 (en) 2007-05-21 2008-11-26 Vivalis Recombinant protein production in avian EBx® cells
EP2215113B1 (en) 2007-10-26 2015-02-25 CSL Limited Cytokine muteins
US8182814B2 (en) * 2007-10-26 2012-05-22 Csl Limited Methods of treating inflammatory airway conditions by inhibition of IL-11 activity
US8518888B2 (en) * 2008-10-14 2013-08-27 Csl Limited Method of treatment of gastrointestinal-type cancer with antagonistic antibodies to IL-11R
AR074369A1 (en) 2008-11-20 2011-01-12 Genentech Inc ANTI-UNC 5B ANTIBODIES (NETRINE RECEIVER) AND METHODS OF USE
KR101940059B1 (en) 2008-12-19 2019-01-18 마크로제닉스, 인크. Covalent diabodies and uses thereof
MX2011007833A (en) 2009-01-23 2011-10-06 Biogen Idec Inc Stabilized fc polypeptides with reduced effector function and methods of use.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Monoclonal antibodies against the human interleukin-11 receptor alpha-chain (IL-11Ralpha) and their use in studies of human mononuclear cells;Blanc C等;《 J Immunol Methods》;20000731;第241卷(第1-2期);43-59页 *

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KR20150115018A (en) 2015-10-13
WO2014121325A1 (en) 2014-08-14
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CN117843785A (en) 2024-04-09
US20140219919A1 (en) 2014-08-07

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